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MX2012005912A - Polypeptide conjugate. - Google Patents

Polypeptide conjugate.

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Publication number
MX2012005912A
MX2012005912A MX2012005912A MX2012005912A MX2012005912A MX 2012005912 A MX2012005912 A MX 2012005912A MX 2012005912 A MX2012005912 A MX 2012005912A MX 2012005912 A MX2012005912 A MX 2012005912A MX 2012005912 A MX2012005912 A MX 2012005912A
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Mexico
Prior art keywords
polypeptide conjugate
compound
further characterized
polypeptide
subject
Prior art date
Application number
MX2012005912A
Other languages
Spanish (es)
Inventor
Soumitra S Ghosh
Behrouz Bruce Forood
James L Trevaskis
Chengzao Sun
Original Assignee
Amylin Pharmaceuticals Inc
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Publication date
Application filed by Amylin Pharmaceuticals Inc filed Critical Amylin Pharmaceuticals Inc
Publication of MX2012005912A publication Critical patent/MX2012005912A/en

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    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
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    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
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    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/643—Albumins, e.g. HSA, BSA, ovalbumin or a Keyhole Limpet Hemocyanin [KHL]
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Abstract

The disclosure provides Polypeptide Conjugates with multiple improved pharmacological and pharmacokinetic properties and their use in treating various diseases and conditions, such as diabetes and/or obesity.

Description

POLYPEPTIDE CONJUGATE INTERREFERENCE WITH RELATED REQUESTS This application claims the benefit of the US provisional patent application. UU No. 61 / 263,752, filed on November 23, 2009, the entire contents of which are incorporated here for all purposes.
FIELD OF THE INVENTION Polypeptide conjugates having both GLP-1 receptor agonist activity and amylin mimetic activity, with superior pharmacological properties, and therapeutic methods for their use are provided herein.
BACKGROUND OF THE INVENTION Peptides and proteins play critical roles in the regulation of biological processes. For example, peptides play a regulatory role as hormones and inhibitors, and are also involved in immunological recognition. The significant biological function of the peptides makes them important to understand their interactions with the receptors to which they bind.
Incretin peptides are peptide hormones and mimetics that cause an increase in the amount of insulin released when glucose levels are normal, or particularly when they are elevated. The concept of the incretin effect was developed from the observation that insulin responses to oral glucose exceed the measurements after intravenous administration of equivalent amounts of glucose. These incretin peptides have other actions beyond the initial action of incretin defined by insulin secretion. For example, they may also take actions to reduce glucagon production, increase satiety or reduce food intake, and delay gastric emptying; In addition, they may have actions to improve insulin sensitivity, and may increase islet cell neogenesis-the formation of new islets.
Although many postprandial hormones have activity similar to incretin, the predominant incretin peptides include the glucose-dependent insulinotropic polypeptide, also known as gastric inhibitory polypeptide (GIP), glucagon-like peptide 1 (GLP-1), and exendin peptides ( which are non-endogenous incretin mimetics). Both GIP and GLP-1 belong to the glucagon peptide superfamily. GLP-1 is secreted by specialized cells in the gastrointestinal tract and has localized receptors on islet cells and also other tissues. GLP-1 is secreted from the intestine in response to the ingestion of nutrients, which results in an increase in insulin secretion. The effect insulinotropic GLP-1 depends on the elevations of the environmental glucose. GLP-1 is rapidly deactivated by the ubiquitous enzyme dipeptidyl peptidase IV (DPP-IV). Exendin 4 binds to GLP-1 receptors on insulin secreting cells, stimulates insulin secretion in the presence of glucose, and the peptide also suppresses glucagon, delays gastric emptying and increases satiety, and reduces ingestion of food. The use of the insulinotropic activities of exendin 4 has been proposed for the treatment of diabetes mellitus and the prevention of hyperglycemia (Eng, U.S. Patent No. 5,424,286). However, unlike GLP-1, exendin 4 has a relatively long half-life in humans, and also has a stronger ability to stimulate insulin secretion at a lower concentration of exendin 4.
Another family of peptide hormones involved in metabolic diseases and disorders is the family of amylin peptide hormones, for example amylin and its analogs, for example pramlintide and davalintide. The amylin molecule has two post-translational modifications: the C-terminus is amidated, and the cysteines at positions 2 and 7 are intertwined to form an N-terminal loop. The sequence of the open reading frame of the human amidine gene shows the presence of the proteolytic cleavage signal of the dibasic amino acid Lys-Arg, before the N-terminal codon for Lys, and the Gly before the proteolytic signal Lys-Arg in the CLAIMS-terminal position, a typical sequence for amidation by the PAM protein amidation enzyme (Cooper et al., Biochem. Biophys Acta 1014: 247-258 (1989)). It is believed that amylin delays gastric emptying and suppresses glucagon secretion, and that it reduces food intake, thus regulating the rate of glucose onset in the circulation. It seems to complement the actions of insulin, which regulates the speed of disappearance of glucose from the circulation and its uptake by peripheral tissues. These actions are supported by experimental findings in rodents and humans, which indicate that amylin complements the effects of insulin in the postprandial control of glucose, at least by three independent mechanisms, all of which affect the rate of appearance of glucose . In human trials, it has been shown that an amylin analog, pramlintide, reduces weight or weight gain. Amylin mimetics may be of benefit in the treatment of metabolic conditions such as diabetes and obesity. Amylin mimetics can also be used to treat pain, bone disorders, gastritis, to modulate lipids, in particular triglycerides, or to affect body composition, such as preferential fat loss and lean tissue saving.
Metabolic diseases and disorders have many forms that include obesity, diabetes, dyslipidemia, insulin resistance, cellular apoptosis, etc. Obesity and its associated disorders are common and very serious public health problems in the United States. UU and all over the world. Higher body obesity is the strongest known risk factor for type 2 diabetes mellitus, and is a strong risk factor for cardiovascular disease. Obesity is a recognized risk factor for diabetes, hypertension, atherosclerosis, congestive heart failure, stroke, gallbladder disease, osteoarthritis, sleep apnea, reproductive disorders such as polycystic ovarian syndrome, breast, prostate and colon cancer, and increased incidence of complications of general anesthesia (see, for example, Kopelman, Nature 404: 635-43 (2000)). Reduces the maximum duration of life and carries a serious risk of the aforementioned comorbidities, as well as disorders such as infections, varicose veins, acanthosis nigricans, eczema, exercise intolerance, insulin resistance, hypercholesterolemia of hypertension, cholelithiasis, damage orthopedic and thromboembolic disease (Rissanen et al., Br. Med. J. 301: 835-7 (1990)). Obesity is also a risk factor for the group of conditions called insulin resistance or "syndrome X". Currently, obesity is a poorly treatable, chronic, essentially intractable metabolic disorder. A therapeutic drug useful in reducing weight of obese or overweight people, with or without diabetes, could have a profound beneficial effect on health.
Diabetes is a disorder of carbohydrate metabolism characterized by hyperglycemia and glucosuria, which result from the production or insufficient use of insulin. Diabetes severely affects the quality of life of large parts of the population of developed countries. Insufficient insulin production is characterized as type 1 diabetes, and insufficient insulin use is type 2 diabetes. However, it is now widely recognized that there are many different diseases related to diabetes, which have their onset well before It is openly diagnosed that patients have diabetes. Also, the effects of suboptimal control of glucose metabolism in diabetes produce a broad spectrum of cardiovascular and lipid-related disorders. Overweight and obesity are a common comorbidity serious with diabetes and in some cases can lead to diabetes or increase the tendency to it.
Dyslipidemia, or abnormal levels of lipoproteins in the blood plasma, is a frequent occurrence among diabetics. Typically, dyslipidemia is characterized by elevated plasma triglycerides, low HDL (low density lipoprotein) cholesterol, normal to high levels of LDL (low density lipoprotein) cholesterol, and increased levels of small dense LDL particles (low density lipoprotein). ) in the blood. Dyslipidemia is one of the main contributors to the increase in the incidence of coronary events and deaths among diabetic subjects. Epidemiological studies have confirmed this, showing a multi-fold increase in coronary deaths among diabetic subjects compared to non-diabetic subjects. Several lipoprotein abnormalities have been described among diabetic subjects.
Attempts to treat the multiple abnormalities associated with diabetes have prompted the administration of several medications antidiabetics to manage these abnormalities in different patients. Examples of antidiabetic drugs are proteins such as insulin and insulin analogs, and small molecules such as insulin sensitizers, insulin secretagogues, and appetite regulating compounds, which are often co-administered to handle the multiple possible abnormalities.
The need to develop useful polypeptides in the diseases, conditions and metabolic disorders described above persists. Accordingly, an object of the present invention is to provide polypeptides useful for treating the diseases and conditions described herein, and methods of producing and using the polypeptides.
BRIEF DESCRIPTION OF THE INVENTION In general terms, the present invention refers to novel polypeptide conjugates with multiple pharmacological actions that allow their use as agents for the treatment and prevention of metabolic diseases and disorders that can be alleviated by controlling plasma glucose levels, of insulin and / or insulin secretion, and / or in addition increasing weight loss, reducing body weight, maintaining weight, preventing weight gain, reducing food intake, increasing satiety, reducing appetite and / or by improving body composition by saving lean mass but fat intake, or by a combination of the glucose / insulin control and weight loss / feed ingestion control properties present in the polypeptide conjugates described herein. Such conditions and diseases include hyperglycemia and conditions related to hyperglycemia, diabetes and conditions related to diabetes, obesity and overweight, and conditions where diseases or conditions related to glucose / insulin control and related to weight loss are present. ingestion of food. In addition, such conditions and disorders include, without limitation, hypertension, dyslipidemia, cardiovascular disease, eating disorders, insulin resistance, obesity and diabetes mellitus of any kind, including type 1, type 2 and gestational diabetes, and combinations from the same; the present multiple action compounds also have use in conditions where hyperglycemia is an important factor, often in the absence of overt diabetes, and also where overweight or obesity are present or may occur, such as in steroid-induced diabetes , diabetes induced by treatment against human immunodeficiency virus (HIV), adult latent autoimmune diabetes (LADA), nonalcoholic steatohepatitis (NASH) and nonalcoholic fatty liver disease (NAFLD), development of diabetes in subjects with congenital lipoatrophy or associated with HIV or "fat redistribution syndrome", and in the metabolic syndrome (syndrome X).
Now it has been unexpectedly discovered that a conjugate of The antidiabetic polypeptide may have similar or improved glucose-lowering effects, and in addition it may have improved effects of weight loss and / or reduction of food intake, as compared to exenatide, ie, exendin 4, a GLP receptor agonist. -1 approved for the treatment of diabetes in the US UU and Europe, and compared to previously known conjugated peptide constructs. The description of the present is based on this discovery. The polypeptide conjugates of the present invention provide at least two pharmacological actions, eg, glucose / insulin control and weight control / feed intake, as described herein, to provide a superior therapeutic benefit, for example by acting in a synergistic way to improve or normalize multiple functions and metabolic abnormalities.
The polypeptide conjugates, compound 1A and compound 2A, described herein, are provided which comprise a GLP-1 receptor agonist peptide conjugated to an amylin mimetic peptide. Polypeptide conjugates exhibit exendin 4 agonism in a GLP-1 receptor and exhibit davalintide agonism (an amylin mimetic) in a C1a receptor to provide, at a minimum, desirable control of glucose and insulin with superior control of weight loss and / or food intake, compared to any exendin 4 or davalintide.
In some embodiments, a polypeptide conjugate described herein is superior to a corresponding reference compound, e.g., exenatide or davalintide, or a reference conjugate compound corresponding having a different GLP-1 receptor agonist component and / or a different amylin mimetic component, for example, compound 3A and compound 7A described herein. In this context, the term "superior" refers to a variety of functional properties that could be weighted in the evaluation of a treatment for a disease or disorder. For example, the polypeptide conjugate described herein may require a lower dose to obtain maximum efficacy, for example 1X, 2X, 3X, 4X, 5X or even less than the corresponding reference compound. For a further example, the polypeptide conjugate described herein could have a higher potency, for example 1.5X, 2X, 3X, 4X, 5X, 10X, 20X, 50X, or even higher potency. For a further example, the polypeptide conjugate described herein could have a function not found to a significant degree in the reference compound.
It is further understood that the polypeptide conjugates described herein include those that have been chemically modified. Such modified peptides include conjugation of one or more polymer portions, such as polyethylene glycol (PEG) or fatty acid chains of various lengths (e.g., stearyl, palmitoyl, octanoyl, etc.), or by the addition of polyamino acids as a poly -his, poly-arg, poly-lys, and poly-ala. Modifications may also include portions of small molecules, such as short alkyls and restricted alkyls (eg, branched, cyclic, fused, adamantyls) and aromatic groups. The polymer portions will normally have a molecular weight of about 500 Dalton to about 60,000 Dalton. The polymer can be linear or branched. Such modifications may occur at the N or C terminus or in a side chain of an amino acid residue, for example the epsilon-amino group of lysine, aspartic acid, glutamic acid, the sulfhydryl group of cysteine, within the polypeptide conjugate. Alternatively, the modification can occur at multiple sites throughout the conjugated polypeptide. To provide a site for modification one or more amino acids can be substituted with, or in addition to, a lysine, aspartic acid, glutamic acid or cysteine; see, for example, U.S. Pat. UU Nos. 5824784 and 5824778, which are incorporated herein by reference.
In one embodiment, the polypeptide conjugates can be conjugated with one, two or three polymer portions. Pegylation of the polypeptide conjugates can improve their aqueous solubility, increase the half-life in the plasma, reduce immunogenicity and / or improve oral intake. In one embodiment, the pegylated polypeptide conjugates comprise a lysine side chain to which it is covalently linked, by the epsilon-amino group of the lysine, a polyethylene glycol moiety, and in a further embodiment the total molecular weight of the PEG is at least about 10,000 Daltons, at least about 20,000 Daltons, at least about 40,000 Daltons or at least 60,000 Daltons. In one embodiment, the molecular weight of the PEG chains is greater than 10,000 Daltons and less than or equal to 60,000 Daltons.
The polypeptide conjugates can be used for therapeutic purposes (for example to treat diabetes); for research purposes; and to produce GLP-1 receptor agonist compounds having enhanced GLP-1 receptor binding activity, and / or improved glucose reduction activity in vivo, and / or improved activity as amylin mimetics, and / or improved weight loss control / feed ingestion activity, such as derivatives having a prolonged duration of action, greater solubility and / or lower immunogenicity. The description provides pharmaceutical compositions comprising therapeutically effective amounts of the polypeptide conjugate. The description also provides methods for synthesizing the polypeptide conjugate.
The description provides methods for the treatment of diabetes; insulin resistance treatment; treatment of hyperglycemia; postprandial decrease in blood glucose levels; decreased HbAlc levels; stimulation of insulin release; reduction of gastric motility; delay of gastric emptying; reduction of food intake; appetite reduction; weight reduction; treatment of overweight; and / or treating obesity in subjects in need thereof, by administering therapeutically effective amounts of the polypeptide conjugate described herein. The description also provides methods for the treatment of hyperglycemia and conditions related to hyperglycemia, diabetes and conditions related to diabetes, obesity and overweight, and conditions where they are present. diseases or conditions related to glucose / insulin control and related to weight loss / food ingestion, in subjects in need thereof, by administering therapeutically effective amounts of a polypeptide conjugate described herein. The description also provides methods for the treatment of hypertension, dyslipidemia, cardiovascular disease, eating disorders, insulin resistance, obesity and diabetes mellitus of any kind, including type 2, type 2 and gestational diabetes, and combinations of same, in subjects in need thereof, administering therapeutically effective amounts of the polypeptide conjugate described herein. The description also provides methods for the treatment of steroid-induced diabetes, diabetes induced by treatment against human immunodeficiency virus (HIV), adult latent autoimmune diabetes (LADA), non-alcoholic steatohepatitis (NASH) and fatty liver disease. non-alcoholic syndrome (NAFLD), development of diabetes in subjects with congenital or HIV-associated lipoatrophy, or "fat redistribution syndrome", and metabolic syndrome (syndrome X), in subjects in need thereof, administering therapeutically effective amounts of the conjugate of polypeptide described herein.
The polypeptide conjugate can be co-administered with another anti-diabetic and / or anti-obesity agent. By "coadministering" is meant the administration of two or more active agents in a single composition, the simultaneous administration in separate solutions, or alternatively they may be administered at different times one with respect to another, such as for example one in the course of , 8 or 12 hours of the other (for example to avoid interference with the taking of the second agent due to the delay of the effects of gastric emptying of the polypeptide conjugate). The exact proportion of the polypeptide conjugate to the second agent will depend in part as determined by the physician and the needs of the subject.
The polypeptide conjugate can be provided in a kit suitable for use by the subject, wherein the kit comprises instructions for using the polypeptide conjugate.
BRIEF DESCRIPTION OF THE FIGURES Figures 1A and 1B: Figure 1A is a graph depicting the change in body weight in DIO rats (with diet-induced obesity) that received compound 2A or the other compounds in the amounts shown in Figure 1A, as described in the examples. Figure 1B is a graph depicting the change in body weight in DIO rats that received Compound 1A or compounds in the amounts shown in Figure 1B, as described in the examples. Groups that do not share a superscript are significantly different from each other; p < 0.05, for example the vehicle controls of Figures 1A and 1B are not significantly different from each other.
Figures 2A and 2B: Figure 2A is a graph depicting the total cumulative feed intake of the DIO rats that received Compound 2A or the other compounds in the amounts shown in Figure 2A, as described in the examples. Figure 2B is a graph representing the total cumulative feed intake of the DIO rats that received compound 1A or the other compounds in the amounts shown in Figure 2B, as described in the examples. Groups that do not share a superscript are significantly different from each other; p < 0.05; for example, the vehicle controls of Figures 2A and 2B are not significantly different from each other.
Figure 3: Figure 3 is a graph showing the change in adiposity in the DIO rats that received Compound 2A or the other compounds in the amounts shown in Figure 3, as described in the examples. Groups that do not share a superscript are significantly different from each other; p < 0.05.
Figure 4: Figure 4 is a graph showing the change in adiposity in DIO rats that received compound 1A or the other compounds in the amounts shown in Figure 4, as described in the examples. Groups that do not share a superscript are significantly different from each other; p < 0.05.
Figure 5: Figure 5 is a graph showing the percentage change in lean mass of the DIO rats that received Compound 2A or the other compounds in the amounts shown in Figure 5, as described in the examples.
Figure 6: Figure 6 is a graph showing the percentage change in lean mass of the DIO rats that received Compound 1A or the other compounds in the amounts shown in Figure 6, as described in the examples; * p < 0.05 against the vehicle.
Figures 7A and 7B. Figures 7A and 7B are graphs showing the measured levels of drug in the plasma (PK property) correlated with the weight loss (PD property) on day 14 (figure 7A) and day 28 (figure 7B) for the compound 1A, compound 2A and compound 3A, in the studies of chronic DIO rats described herein.
DETAILED DESCRIPTION OF THE INVENTION "GLP-1 receptor agonist compounds" refer to compounds that elicit a biological activity of an exendin reference peptide (e.g., exendin 4) or a GLP-1 reference peptide (7-37), when evaluated by known measurements, such as receptor binding studies, cAMP generation, or in vivo tests of blood glucose and / or insulin secretion, as described herein, and by means of Hargrove et al, Regulatory Peptides , 141: 113-1 19 (2007), the description of which is incorporated herein by reference. GLP-1 receptor agonist compounds include, for example, native exendins, exendin analogs, native GLP-1, GLP-1 analogs, GLP-1 (7-37), and GLP-1 analogs (7- 37).
The term "exendin" includes natural exendin peptides (or synthetic versions of the natural ones) found in the salivary secretions of the Gila monster. Exendins include the amidated forms, the acid form, the pharmaceutically acceptable salt form, and any other physiologically active form of the molecule. In one embodiment, the term "exendin" can be used interchangeably with the term "exendin agonist".
The "exendin analogue" refers to peptides, peptides that contain substitutions, insertions, deletions or amino acid additions, peptide mimetics and / or other modifications, and / or other chemical moieties, which cause a biological activity similar to that of a exendin reference peptide (eg, exendin 4), when evaluated by known measurements, such as the receptor binding tests or the in vivo blood glucose assays described herein and in Hargrove et al. , Regulatory Peptides, 141: 113-1 19 (2007). Exendin analogs include the amidated forms, the acid form, the pharmaceutically acceptable salt form, and any other physiologically active form of the molecule. In one embodiment, the term "exendin analog" can be used interchangeably with the term "exendin agonist analog".
Exenatide (exendin 4) is a glucagon-like peptide receptor agonist (GLP-1) of 39 amino acids, currently indicated for the treatment of type 2 diabetes, and also exerts weight loss actions and other metabolic actions when administered to DIO rats. The sequence of exendin 4 is as follows: HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS-NH2 (SEQ ID NO: 1), wherein -NH2 indicates the presence of a C-terminal amidated amino acid. Exendin 4 is also active in its free acid form. Hargrove et al, Regulatory Peptides, 141: 1 13-119 (2007), reported a peptide analogue of exendin 4 which is a C-terminally full-length amidelated peptide of exendin 4 peptide, with a single nucleotide difference in the position 14 compared to the native exendin 4, and which is referred to herein as the compound 4A. Hargrove reported that this exendin 4 analog, compound 4A, is markedly inferior to exendin 4 with respect to its delayed gastric emptying, anti-obesity properties and half-life. The sequence of compound 4A is as follows: HGEGTFTSDLSKQLEEEAVRLFIEWLKNGGPSSGAPPPS-NH2 (SEQ ID NO: 2). Another peptide analog of exendin 4 is referred to herein as compound 5, which is a chimera of the first 32 amino acids of exendin 4 having amino acid substitutions at positions 14 and 28, followed by a sequence of 5 amino acids of the C-terminus of a GLP-1 different from mammal (frog). Compound 5 has the following sequence: HGEGTFTSDLSKQLEEEAVRLFIEWLKQGGPSKEIIS (SEQ ID NO: 3).
A truncated, biologically active form of exendin 4, exendin 4 (1-28) is also known, which is referred to herein as compound 10 and has the following sequence: HGEGTFTSDLSKQMEEEAVRLFIEWLKN (SEQ.
ID NO: 4); and its amide form is called compound 10A and has the sequence HGEGTFTSDLSKQMEEEAVRLFIEWLKN-NH2 (SEQ ID NO: 5).
Amylin is a peptide hormone co-secreted with insulin by pancreatic beta cells after ingestion of nutrients, whose primary physiological functions include inhibition of food seeking behavior and gastric emptying, and subsequently reduces body weight. Davalintide, also known as "AC-2307," is an amylin agonist that has been reported to be useful in the treatment of a variety of pathological indications; see WO 2006/083254 and WO 2007/14838, each of which is incorporated herein by reference in its entirety and for all purposes. Davalintide is a chimeric peptide having an N-terminal loop region of amylin or calcitonin and analogs thereof, an alpha-helical region of at least a portion of an alpha-helical region of calcitonin or analogues thereof, or an alpha-helical region having a portion of an alpha-helical region of amylin and an alpha-helical region of calcitonin or analogue thereof, and a C-terminal tail region of amylin or calcitonin. The amino acid sequence of davalintide is as follows: KCNTATCVLGRLSQELHRLQTYPRTNTGSNTY-NH2 (SEQ ID NO: 6), which is referred to herein as compound 6A. It has been reported that for rats with diet-induced obesity (DIO), the weight loss induced by amylin and by the administration of compound 6A is preferably characterized by loss of adipose mass and preservation of lean mass.
The previously described conjugates of an exendin analog and an amylin mimetic include compound 3A having the sequence: HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGGKCNTATCVLGRL SQELHRLQTYPRTNTGSNTY-NH2 (SEQ ID NO: 7), wherein the polypeptide is C-terminally amidated, and compound 7A which has the sequence: HGEGTFTSDLSKQMEEEAVRLFIEWLKN (beta-A) (beta-A) KCNTATCVLGR LSQELHRLQTYPRTNTGSNTY-NH2 (SEQ ID NO: 8), wherein the polypeptide is C-terminally amidated and contains an unnatural amino acid dipeptide linker of beta-alanine , beta-alanine. The above compounds comprise a C-terminally truncated active form of exendin 4, compound 10A exendin 4 (1-28) amide: HGEGTFTSDLSKQMEEEAVRLFIEWLKN-NH2 (SEQ ID NO: 5).
The present invention is based on the surprising result of the superior effects of specific polypeptide conjugates described herein that comprise exendin-4-specific peptide analogs covalently linked to davalintide, compound 6A. The polypeptide conjugates are compound 1A and compound 2A described herein and their chemical derivatives, for example pegylated. The polypeptide conjugates exhibit exendin 4 agonism at the GLP-1 receptor and davalintide agonism (an amylin mimetic) at the C1a receptor to, at least, provide glucose control with superior weight loss control. food ingestion compared to exendin 4 or davalintide. The compounds produce superior glucose control with surprisingly superior weight control, and also superior pharmacokinetic properties compared to the known conjugates and the origin peptides. The polypeptide conjugates have a combination of glucose-regulator GLP-1 receptor agonism and weight loss inducer, with the amylin mimetic activity inducing fat-specific weight loss, but with superior properties, for example higher therapeutic efficacy, life improved media, with respect to the origin peptide or the known conjugates of this type.
The term "polypeptide conjugate" with respect to the present invention refers to compound 1A and compound 2A, which are C-terminally amidated and include their acid form, their pharmaceutically acceptable salt forms and any other physiologically active form of compound 1a or compound 2A, including chemically modified derivatives, for example by modification of pegylation or acylation of fatty acid, in order to extend the half-life in plasma or improve oral intake.
The compound 1 described herein has the following sequence: HGEGTFTSDLSKQLEEEAVRLFIEWLKNGGPSSGAPPPSGGGKC NTATCVLGRLSQELHRLQTYPRTNTGSNTY (SEQ ID NO: 9). Its C-terminally amidated form is compound 1A, a polypeptide conjugate of the present invention having the sequence HGEGTFTSDLSKQLEEEAVRLFIEWLKNGGPSSGAPPPSGGGKCNTATCVL GRLSQELHRLQTYPRTNTGSNTY-NH2 (SEQ ID NO: 10). Compound 1A is a polypeptide conjugate of compound 4A covalently linked in frame to compound 6A by means of a glycine-glycine-glycine peptide linker. Compound 2 has the following sequence HGEGTFTSDLSKQLEEEAVRLFIEWLKQGGPSKEIISGGGKCNTATCVLGRL SQELHRLQTYPRTNTGSNTY (SEQ ID NO: 11). Its C-terminally amidated form is compound 2A, a polypeptide conjugate of the present invention having the sequence HGEGTFTSDLSKQLEEEAVRLFIEWLKQGGPSKEIISGGGKCNTATCVLGRL SQELHRLQTYPRTNTGSNTY-NH2 (SEQ ID NO: 12). Compound 2A is a polypeptide conjugate of compound 5 covalently linked in frame to compound 6A by means of a glycine-glycine-glycine peptide linker.
The present study has characterized the metabolic and PK actions of compound 1A and compound 2A, comprising exendin 4 analogs covalently linked to amylin mimetic davalintide (compound 6A). As described in the examples, the effect of 4 weeks of constant subcutaneous infusion of compound 1A and compound 2A (at 3, 10, 30 and 100 nmol / kg / d) were compared with the individual administration and the co-administration of the peptides of origin, compound 6A, compound 5 and compound 4A (at 2.8, 15 and 7.2 nmol / kg /, maximum effective dose for weight loss) in male Sprague Dawley rats with diet-induced obesity (DIO). In summary, compound 1A and compound 2A were more effective for weight loss with respect to the individual administration of the origin peptides in DIO rats, compound 1A exhibiting greater potency and efficacy for body weight loss in comparison with compound 2A. Compound 1A and compound 2A reduced, in a dose-dependent manner, body weight compared to vehicle controls. The body weight loss induced by compound 1A or compound 2A was associated with a significantly decreased percentage of fat mass (at the dose of 100 nmol / kg / d: -12.2 ± 1.3% for compound 2A, and -15.5 ± 2.2% for compound 1A, both p < 0.05, against vehicle controls). Compound 2A does not alter the percentage of lean mass, however all doses of Compound 1A improved body composition by increasing the percentage of lean mass with respect to vehicle controls. At the highest dose tested, the total food intake was significantly reduced by compound 1A and compound 2A with respect to the individual administration of the source peptide. At the highest dose of compound 1A, the food ingestion was suppressed more than the co-administration of the compounds of origin, compound 4A and compound 6A. After 28 days there was no change in glucose, percentage of hemoglobin A1c (HbA1c), insulin, total cholesterol or HDL, with the treatment with compound 2A. Similarly, there was no significant effect of compound 1A on total cholesterol or HDL or HbA1c levels after 28 days. However, insulin and glucose levels in the plasma were significantly reduced by compound 1A at some doses, compared to vehicle controls. The triglycerides in the plasma were significantly reduced by compound 1A and compound 2A compared to the vehicle after 28 days of treatment. The plasma levels of compound 1A and compound 2A, measured by means of a specific immunoassay, were increasing and corresponding to the treatment dose after 2 and 4 weeks of treatment.
Both compound 1A and compound 2A induced significant body weight loss in a dose-dependent manner., at the highest doses tested, exceeded the weight loss induced by maximum effective doses of the source hormones administered as a single peptide treatment. The weight loss caused by the highest dose of each peptide conjugate (100 nmol / kg / d) was close to 30% (corrected per vehicle) and was the same (eg compound 2A) or higher (e.g. 1A) than the weight loss achieved by coinfusion of the origin peptides. Previous reference conjugates, for example compound 3A and compound 7A, were significantly less potent in reducing body weight than the compounds of origin despite greater efficacy. In surprising contrast, compound 1A and compound 2A showed a higher potency than compound 3A and compound 7A. Meanwhile only a weight loss of -20% was achieved with 100 nmol / kg / d of compound 3A and compound 7A; Similar weight loss was achieved with only 10 nmol / kg / d of compound 1A and compound 2A. In addition, the lower dose of 3 nmol / kg / d of compound 1A or compound 2A, caused a significant body weight loss of ~ 11-19%, compared to vehicle controls, during the 4 week period, of similarly to compound 6A (12%), compound 4A (14%) and compound 5 (17%). Compound 1A was also more effective than compound 2A for weight loss (-37% vs. -28%, respectively).
The body weight loss induced by compound 1A and compound 2A was associated with dose-dependent reductions in food intake. Additionally, treatment with compound 1A was associated with reduction of fat mass and concomitant preservation of lean mass, an improvement in body composition. Although the control DIO rats were not hyperinsulinemic or diabetic, modest effects on insulin reduction were observed, but unexpectedly no glucose or HbA1c was observed with compound 1A, as well as with the single administration or coadministration of the origin peptide.
Plasma levels of compound 1A and compound 2A were measured on day 14 and day 28 of the study in the rats described in the examples, using a specific assay. Although approximately dose-dependent increases were observed in the two polypeptide conjugates, the levels of Compound 2A appeared much higher than those of Compound 1A, especially after 28 days. However, when the PK profile was aligned with the pharmacological profile (PD) of each compound, the PD effect of compound 2A was not extended to match its PK profile. In other words, although compound 2A could be accumulating in serum during the study period, its PD effect had decreased in comparison with compound 1A. Without wishing to be bound by theory, this discrepancy may represent an early development of antibodies to compound 2A. Although this difference was due to some interference (plasma ab) in the PK test of compound 2A, the concomitant extended PD and PK profiles of compound 1A in comparison provide another important, surprisingly superior property of compound 1A over compound 2A. In general, compound 1A and compound 2A exerted a remarkable loss of fat specific weight in DIO rats, superior to compound 3A, compound 1A being superior to the other two compounds. The treatment of compound 1A, which has a very high degree of potency and efficacy, also showed improvements of other metabolic parameters including triglyceride reduction and antidiabetic actions, as well as absence or reduction of nausea compared to reference compounds and conjugates (see the examples).
In one embodiment, the polypeptide conjugates described herein include those that have been chemically modified. Such modified polypeptide conjugates include conjugation with one or more polymer portions, such as polyethylene glycol (PEG) or amino acid chains of various lengths (eg, stearyl, palmitoyl, octanoyl, etc.), or by the addition of polyamino acids such as poly-his, poly-arg, poly-lys, and poly-ala. Modifications may also include portions of small molecules such as short alkyls and restricted alkyls (eg branched, cyclic, fused, adamantyls), and aromatic groups.
The polymer portions will normally have a molecular weight of about 500 Dalton to about 60,000 Dalton. The polymer can be linear or branched. Such modifications may occur at the N or C terminus or on a side chain of an amino acid residue, for example, the epsilon-amino group of the Usin, aspartic acid, glutamic acid, the sulfhydryl group of cysteine, within the polypeptide conjugate. . Alternatively, the modification can occur at multiple sites throughout the conjugated polypeptide. To provide a site for modification, one or more amino acids can be substituted with, in addition to, a lysine, aspartic acid, glutamic acid or cysteine; see, for example, U.S. Pat. UU Nos. 5824784 and 5824778, which are incorporated herein by reference.
In one embodiment, the polypeptide conjugates can be conjugated with one, two or three polymer portions. In one embodiment, the compounds are linked with a polyethylene glycol. Pegylation of the polypeptide conjugates can improve their aqueous solubility, increase the half-life in the plasma, reduce immunogenicity and / or improve oral intake. Polyethylene glycol can have a molecular weight of about 200 Daltons to about 80,000 Daltons; from about 5,000 Dalton to about 60,000 Daltons; from about 10,000 Dalton to about 50,000 Daltons; or from approximately 15,000 Dalton to approximately 40,000 Dalton. The polyethylene glycol may be linear or branched. In one embodiment, the pegylated polypeptide conjugates they comprise a lysine side chain to which it is covalently linked, by the epsilon-amino group of lysine, a polyethylene glycol moiety. In a further embodiment, the total molecular weight of the PEG portion is at least about 10,000 Daltons, at least about 20,000 Daltons, at least about 40,000 Daltons or at least about 60,000 Daltons.
In one embodiment, the compounds are linked to one or two polyethylene glycols, wherein the polyethylene glycol is further bonded to a lipophilic moiety. In one embodiment, polyethylene glycol in this case can have a molecular weight of about 200 Dalton, to about 7,000 Dalton, or about 500 Dalton to about 5,000 Dalton. The lipophilic moiety may be an alkyl group (eg, an alkyl group of C 20, a C 0 alkyl group, an alkyl group of Ci-β, an alkyl group of C-), a fatty acid (e.g. of fatty acid of C4-28 ', a fatty acid chain of Ce-24, a fatty acid chain of C10-20), cholesteryl, adamantyl, and the like. The alkyl group can be linear or branched, preferably linear. In one embodiment, the fatty acid is an acetylated fatty acid or an esterified fatty acid. The lipophilic portion - (polyethylene glycol) - may be linked to the compound at the C-terminal amino acid residue, an N-terminal amino acid residue, an internal amino acid residue (for example an internal Lys amino acid residue), or a combination thereof. the same (for example, the compound binds at the N-terminal and C-terminal amino acid residues). In said embodiments, the derivative has superior oral uptake and bioavailability as compared to unmodified compound 1A or compound 2A.
In one embodiment, the compounds are linked to a polyamino acid. Exemplary polyamino acids include poly-lysine, poly-aspartic acid, poly-serine, poly-glutamic acid, etc. The polyamino acid may be in the D or L form, preferably in the L form. The polyamino acids may comprise from 1 to 12 amino acid residues; from 2 to 10 amino acid residues; or from 2 to 6 amino acid residues. The derivative can provide a longer duration of action compared to the unmodified compound 1A or compound 2A.
In one embodiment, the compounds are linked with a fatty acid. The fatty acid may be a fatty acid chain of C4.28, a fatty acid chain of Ce-24, or a fatty acid chain of Cio-2o- In one embodiment, the fatty acid is an acetylated fatty acid. In one embodiment, the fatty acid is an esterified fatty acid. The derivative can provide a longer duration of action compared to the unmodified compound 1A or compound 2A.
In one embodiment, the compounds bind to albumin. The albumin can be a recombinant albumin, serum albumin or recombinant serum albumin. In another embodiment, the compounds are linked to an albumin-fatty acid (i.e., an albumin bound to a fatty acid). The derivative can provide a longer duration of action compared to the unmodified compound 1A or compound 2A.
In one embodiment, the compounds are linked to an immunoglobulin or an immunoglobulin Fe region. The immunoglobulin can be IgG, IgE, IgA, IgD, or IgM. In one embodiment, the compounds are linked to an Fe region of IgG or an Fe region of IgM. The immunoglobulin Fe region is (i) the heavy chain constant region 2 (CH2) of an immunoglobulin; (I) the heavy chain constant region 3 (CH3) of an immunoglobulin; or (iii) the heavy chain 2 (CH2) and 3 (CH3) constant regions of an immunoglobulin. In addition, the immunoglobulin Fe region can comprise the hinge region in the heavy chain constant region. Other embodiments for the immunoglobulin Fe region that can be linked to the exendin analog peptides are described in WO 2008/082274, the disclosure of which is incorporated herein by reference. The derivative can provide a longer duration of action compared to the unmodified compound 1A or compound 2A.
When the polypeptide conjugates described herein are covalently linked to one or more polymers as described herein, any known linker group can be used. The linker group can comprise any suitable chemical group for linking the peptide to the polymer. Alternatively, the polypeptide conjugates can be attached directly to the polymer without any linking group. Exemplary linker groups include amino acids, maleimido groups, dicarboxylic acid groups, succinimide groups, or a combination of two or more thereof. Methods for linking peptides to one or more polymers are known and described for example in U.S. Pat. UU No. 6,329,336; the US patent UU No. 6,423,685; the US patent UU No. 6,924,264; WO 2005/077072, WO 2007/022123, WO 2007/053946; WO 2008/058461; and WO 2008/082274, the descriptions of which are incorporated herein by reference.
The administered polypeptide conjugate may be in the form of a prodrug. The term "prodrug" refers to a compound that is a drug precursor which, after administration, releases the drug in vivo by some chemical or physiological process, for example proteolytic cleavage, or after reaching a medium of a certain pH.
The polypeptide conjugates described herein can be prepared using biological, chemical, and / or recombinant DNA techniques that are known. Exemplary methods are described in U.S. Pat. UU No. 6,872,700; WO 2007/139941; WO 2007/140284; WO 2008/082274; WO 2009/011544; and US publication UU No. 2007/0238669, whose descriptions are incorporated herein by reference. Other methods for preparing the polypeptide conjugates are described herein.
The polypeptide conjugates described herein can be prepared using standard solid phase peptide synthesis techniques, such as an automatic or semi-automatic peptide synthesizer. Typically, using such techniques, a protected alpha-N-carbamoyl amino acid and an amino acid added to the growing peptide chain on a resin are coupled at room temperature in an inert solvent (eg dimethylformamide, N-methylpyrrolidinone, methylene chloride, etc.), in the presence of coupling agents (eg, dicyclohexylcarbodiimide, 1-hydroxybenzotriazole, etc.), in the presence of a base (eg, diisopropylethylamine, etc.). The alpha-N-carbamoyl protecting group is removed from the resulting peptide-resin using a reagent (for example trifluoroacetic acid, piperidine, etc.), and the coupling reaction is repeated with the next desired N-protected amino acid to be added to the peptide chain. Suitable N-protecting groups are well known, such as t-butyloxycarbonyl (tBoc), fluorenylmethoxycarbonyl (Fmoc), and the like. The solvents, amino acid derivatives and the 4-methylbenzohydrylamine resin used in the peptide synthesizer can be purchased from Applied Biosystems Inc. (Foster City, California).
For the chemical synthesis of the polypeptide conjugates, solid phase peptide synthesis can be used, since in general the solid phase synthesis is a direct approach with excellent scalability up to the commercial scale, and is generally compatible with relatively high polypeptide conjugates. big. Peptide synthesis in solid phase can be performed with an automatic peptide synthesizer (Model 430A, Applied Biosystems Inc., Foster City, California), using the NMP / HOBt system (Option 1), and tBoc or Fmoc chemistry (see The Applied Biosystems "User's Manual" for the ABI 430A peptide synthesizer, Version 1.3B, July 1, 1988, section 6, p. 49-70, Applied Biosystems, Inc., Foster City, California) with blocking. The Boc-peptide-resins can be divided with HF (-5 ° C at 0 ° C, one hour). The peptide can be extracted from the resin by alternating water and acetic acid, and the filtrates are lyophilized. The Fmoc-peptide-resins can be divided according to standard methods (for example, "Introduction to Cleavage Techniques", Applied Biosystems, Inc., 1990, pp. 6-12). Peptides can also be assembled using an Advanced Chem Tech Synthesizer synthesizer (Model MPS 350, Louisville, Kentucky).
The peptides can be purified by RP-HPLC (preparative and analytical) using a Waters Delta Prep 3000 system. A C4, C8 or C18 preparative column of 10μ, 2.2X25 cm can be used; Vydac, Hesperia, California) to isolate the peptides, and the purity can be determined using a C4, C8 or C18 analytical column (5μ, 0.46X25 cm; Vydac). The solvents (A = 0.1% TFA / water, and B = 0.1% TFA / CH3CN) can be supplied to the analytical column at a flow rate of 1.0 ml / min and to the preparative column at 15 ml / min. The amino acid analysis can be done with the Waters PicoTag system and can be processed using the Maximum program. The peptides can be hydrolyzed by acid hydrolysis in the vapor phase (1 15 ° C, 20-24 h). Hydrolysates can be modified and analyzed by standard methods (Cohen et al, "The Pico Tag Method: A Manual of Advanced Techniques for Amino Acid Analysis", pp. 1-52, Millipore Corporation, Milford, Massachusetts (1989)) . A rapid atom bombardment analysis can be done by means of M-Scan, Incorporated (West Chester, Pa.). The mass calibration can be done using cesium iodide or cesium iodide / glycerol. Plasma desorption ionization analysis can be done using time-of-flight detection in an Applied Biosystems Bio-lon 20 mass spectrometer.
Non-peptide polypeptide conjugates can be prepared by known methods. For example, phosphate-containing amino acids and peptides containing such amino acids can be prepared using the known methods, as described in Bartlett er al, Biorg. Chem., 14: 356-377 (1986).
Alternatively, polypeptide conjugates can be produced by known recombinant techniques; see for example Sambrook et al, "Molecular Cloning: A Laboratory Manual", 2nd ed., Cold Spring Harbor (1989). These polypeptide conjugates produced by recombinant technology can be expressed starting from a polynucleotide. The person skilled in the art will appreciate that the polynucleotides, including DNA and RNA, which encode such polypeptide conjugates, can be obtained from wild-type cDNA, for example from exendin 4, amylin, taking into account the degeneracy of the use of code, and can also be built by engineering at will to incorporate the indicated substitutions. These polynucleotide sequences can incorporate codons that facilitate the transcription and translation of mRNA in microbial hosts. Such manufactured sequences can be easily constructed according to known methods; see for example WO 83/04053. Optionally, the above-mentioned polynucleotides can also encode an N-terminal methionyl residue. Non-peptide polypeptide conjugates useful in the present invention can be prepared by known methods. For example, phosphate-containing amino acids and peptides containing said amino acids can be prepared using the known methods; see for example Bartlett and Landen, Bioorg. Chem. 14: 356-77 (1986).
A variety of vector / host expression systems can be used to contain or express a polypeptide conjugate coding sequence. These include, without limitation, microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid or cosmid DNA expression vectors; yeast transformed with expression vectors in yeast; insect cell system infected with virus expression vectors (eg baculovirus); plant cell systems transfected with virus expression vectors (eg, cauliflower mosaic virus, CaMV, tobacco mosaic virus, TMV), or transformed with bacterial expression vectors (eg, the Ti plasmid or pBR322); or animal cell systems. Mammalian cells that are useful in recombinant protein productions include, without limitation, VERO cells, HeLa cells, Chinese hamster ovary cell lines (CHO), COS cells (such as COS-7), Wl 38 cells, BHK , HepG2, 3T3, RIN, MDCK, A549, PC12, K562 and 293. Exemplary protocols are described herein for the recombinant expression of the protein.
Therefore, the polynucleotide sequences are useful for generating new and useful plasmid and viral DNA vectors, new and useful transformed and transfected prokaryotic and transkary host cells (including bacterial, yeast and mammalian cells grown in culture), and methods new and useful for the cultured growth of said host cells, capable of expressing the present conjugated polypeptides. The polynucleotide sequences encoding the polypeptide conjugates herein may be useful for gene therapy in cases where the underproduction of the polypeptide conjugates is alleviated, or the need for higher levels of such conjugates is met.
The present invention also provides methods for the production of recombinant DNA of the conjugated polypeptides of the present invention. A method is provided for producing the polypeptide conjugates from a host cell containing nucleic acids encoding said polypeptide conjugate, comprising: (a) culturing said host cell containing polynucleotides encoding said polypeptide conjugate, under conditions that facilitate the expression of said DNA molecule; and (b) obtaining said conjugated polypeptide.
Host cells can be prokaryotic or eukaryotic and include bacteria, mammalian cells (such as Chinese hamster ovary (CHO) cells, monkey cells, baby hamster kidney cells, cancer cells or other cells), yeast cells and insect cells.
The mammalian host systems for expression of the Recombinant protein are also well known to those skilled in the art. Host cell strains can be chosen for a particular ability to process the expressed protein or produce certain post-translational modifications that will be useful in providing the activity of the protein. Such polypeptide modifications include, without limitation, acetylation, carboxylation, glycosylation, phosphorylation, lipidation and acylation. Post-translation processing, which cuts a "prepro" form of the protein, may also be important to correct insertion, folding and / or function. Different host cells, such as CHO, HeLa, MDCK, 293, WI38, etc., have specific cellular machinery and characteristic mechanisms for said post-translational activities, and can be chosen to ensure the correct modification and processing of the foreign protein introduced.
Alternatively, a yeast system can be used to generate the polypeptide conjugates of the present invention. The coding region of the DNA of the polypeptide conjugates is amplified by means of PCR. A DNA encoding the pre-pro-alpha leader sequence is amplified from yeast genomic DNA in a PCR reaction, using an primer containing nucleotides 1-20 of the alpha equalization factor gene and another primer complementary to the nucleotides 255-235 of this gene (Kurjan and Herskowitz, Cell, 30: 933-43 (1982)). The pre-pro-alpha leader coding sequence and the coding sequence fragments of the polypeptide conjugate are ligated into a plasmid containing the alcohol promoter yeast dehydrogenase (ADH2), such that the promoter directs the expression of a fusion protein consisting of the pre-pro-alpha factor fused to the mature conjugated polypeptide. As Rose and Broach teach, Meth. Enz. 185: 234-79, Goeddel ed., Academic Press, Inc., San Diego, California (1990), the vector also includes an ADH2 transcription terminator in front of the cloning site, the "2-micron" origin of replication of yeast , the yeast leu-2d gene, the REP1 and REP2 genes of yeast, the beta-lactamase gene of E. coli, and an origin of replication of E. coli. The beta-lactamase and leu-2d genes provide selection in bacteria and yeast, respectively. The gene leu-2d also facilitates increasing the number of plasmid copies in the yeast to induce a higher degree of expression. The REP 1 and REP2 genes encode proteins involved in the regulation of the copy number of the plasmid.
The DNA construct described in the preceding paragraph is transformed into yeast cells using a known method, for example treatment with lithium acetate (Stearns et al., Meth., 185: 280-97 (1990)). The ADH2 promoter is induced by glucose depletion in the growth medium (Price ef al., Gene 55: 287 (1987)). The pre-pro-alpha sequence effects the secretion of the fusion protein from the cells. Concomitantly, the yeast KEX2 protein cuts the pre-pro sequence of the mature polypeptide conjugates (Bitter et al., Proc. Nati, Acad. Sci. USA 81: 5330-4 (1984)).
The polypeptide conjugates of the invention are also they can express recombinantly in yeast, for example Pichia, using a commercially available expression system, for example the Pichia Expression System expression system (Invitrogen, San Diego, California), following the manufacturer's instructions. This system is also based on the pre-pro-alpha sequence to direct secretion, but transcription of the insert is driven by the alcohol oxidase (AOXI) promoter by induction with methanol. The secreted polypeptide conjugate is purified from yeast growth medium, for example by the methods used to purify said polypeptide conjugate from bacterial supernatants and from mammalian cells.
Alternatively, the DNA encoding a polypeptide conjugate can be cloned into a baculovirus expression vector, for example pVL1393 (PharMingen, San Diego, California). This vector encoding the conjugated polypeptide is then used according to the manufacturer's instructions (PharMingen) or the known techniques for infecting Spodoptera frugiperda cells, developed for example in protein-free sF9 medium, and producing recombinant protein. The protein is purified and concentrated from the medium using the known methods, for example a heparin Sepharose column (Pharmacia, Piscataway, New Jersey), and sequential molecular sizing columns (Amicon, Beverly, Massachusetts), and re-suspended in a appropriate solution, for example PBS. An analysis of SDS-PAGE can be used to characterize the protein, for example by showing a single band that confirms the size of the desired conjugate protein, as well as the analysis of the complete amino acid sequence, for example Edman sequencing in a sequencer of Proton 2090 peptides, or confirmation of their N-terminal sequence.
For example, the DNA sequence encoding the predicted mature polypeptide conjugate can be cloned into a plasmid containing a desired promoter, and optionally a leader sequence (see for example Better ef al., Science 240: 1041-3 (1988)). ). The sequence of this construction can be confirmed by automatic sequencing. The plasmid is then transformed into E. coli, strain MC1061, using standard procedures using incubation with CaCl2 and heat shock treatment of the bacteria (Sambrook et al., Supra). The transformed bacteria are grown in LB medium supplemented with carbenicillin, and the production of the expressed protein is induced by growth in a suitable medium. If present, the leader sequence will affect the secretion of the mature polypeptide conjugate and will be cut off during secretion. The secreted recombinant polypeptide conjugate is purified from the bacterial culture medium by the method described herein.
Alternatively, polypeptide conjugates can be expressed in an insect system. Insect systems for protein expression are well known to those skilled in the art. In one such system, Autographa Californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes in Spodoptera frugiperda cells or in Trichoplusia larvae. The coding sequence of the polypeptide conjugate is cloned into a non-essential region of the virus, such as the polyhedrin gene, and placed under the control of the polyhedrin promoter. Successful insertion of a polypeptide conjugate will render the polyhedrin gene inactive and produce recombinant virus lacking the protein coat. Then, the recombinant viruses are used to infect S. frugiperda cells or Trichoplusia larvae, in which the polypeptide conjugate of the present invention is expressed (Smith et al., J. Virol 46: 584 (1983); Engelhard et al., Proc. Nati Acad. Sci. USA 91: 3224-7 (1994)).
In another example, the DNA sequence encoding the polypeptide conjugates can be amplified by PCR and can be cloned into an appropriate vector, for example pGEX-3X (Pharmacia, Piscattaway, New Jersey). The pGEX vector is designed to produce a fusion protein comprising glutathione-S-transferase (GTS), encoded by the vector, and a protein encoded by a DNA fragment inserted into the cloning site of the vector. PCR primers can be generated to include, for example, an appropriate cleavage site. Then, the recombinant fusion protein can be cut from the GST portion of the fusion protein. The pGEX-3X construct / polypeptide conjugate is transformed into E. coli XL-1 Blue cells (Stratagene, La Jolla, California), and the individual transformants are isolated and grown at 37 ° C in LB medium (supplemented with carbenicillin ), at an optical density of 0.4 at a wavelength of 600 mm, followed by further incubation for 4 hours in the presence of 0.5 mM isopropyl-beta-D-thiogalactopyranoside (Sigma) Chemical Co., San Luis, Missouri). The plasmid DNA of the individual transformants is purified, and their partial sequence is determined using an automatic sequencer to confirm the presence of the insert of the gene encoding the desired conjugated polypeptide in the proper orientation.
The fusion protein, when it is desired to produce as an inclusion body insoluble in bacteria, can be purified in the following manner. The cells are harvested by centrifugation. They are washed in 0.15 M NaCl, 10 mM Tris, pH 8, 1 mMl EDTA and treated with lysozyme, 0.1 mg / ml (Sigma Chemical Co) for 15 minutes at room temperature. The lysate is clarified by sonication and the cell debris is pelleted by centrifugation for 10 minutes at 12,000 x g. The pellet containing the fusion protein is resuspended in 50 mM TRIS, pH 8, and 10 mM EDTA, deposited as a layer on 50% glycerol, and centrifuged for 30 minutes at 6000 x g. The pellet is resuspended in standard phosphate buffered saline (PBS) free of Mg ++ and Ca ++. The fusion protein is further purified by fractionating the resuspended pellet on a denaturing SDS polyacrylamide gel (Sambrook et al., Supra). The gel is soaked in 0.4 M KCI to visualize the protein, which is cut and electronically eluted in gel run buffer that lacks SDS. If the GST / conjugated polypeptide fusion protein is produced in the bacterium as a soluble protein, it can be purified using the GST purification module (Pharmacia Biotech).
The fusion protein can be digested to cut the GST of the mature conjugate polypeptide. The digestion reaction (20-40 pg of fusion protein, 20-30 units of human thrombin (4000 U / mg (Sigma) in 0.5 ml of PBS) is incubated 16-48 hours at room temperature and loaded onto a gel of denaturing SDS-PAGE to fractionate the reaction products The gel is soaked in 0.4 M KCI to visualize protein bands The identity of the protein band corresponding to the expected molecular weight of the polypeptide conjugate can be confirmed by analysis of partial amino acid sequence using an automatic sequencer (Applied Biosystems Model 473A, Foster City, California).
In a particularly exemplary method of recombinant expression of the polypeptide conjugates of the present invention, 293 cells can be co-transfected with plasmids containing cDNAs of the polypeptide conjugates into the pCMV vector (5'CMV promoter, 3'HGH poly-A sequence ) and pSV2neo (containing the neo resistance gene), by means of the calcium phosphate method. In one embodiment, the vectors must be linearized with Seal before transfection. Similarly, an alternative construct can be used using a similar pCMV vector with the incorporated neo gene. Stable cell lines are selected from individual cell clones by limiting dilution in growth medium containing 0.5 mg / ml of G418 (antibiotic similar to neomycin) for 10-14 days. The cell lines are selected by the expression of the polypeptide conjugates by ELISA or Western blot, and the high expression cell lines are expanded for large scale growth.
It is preferable to use the transformed cells for long-term high-yield protein production, and therefore stable expression is desirable. Once said cells are transformed with vectors containing selectable markers together with the desired expression cassette, the cells can be allowed to grow for 1-2 days in an enriched medium before being switched to a selective medium. The selectable marker is designed to confer resistance to selection, and its presence allows the growth and recovery of cells that successfully express the introduced sequences. Resistant clusters of stably transformed cells can be proliferated using cell culture techniques appropriate for the cell.
Various selection systems can be used to recover the cells that have been transformed for the production of recombinant protein. Such selection systems include, without limitation, the HSV genes thymidine kinase, hypoxanthine-guanine phosphoribosyltransferase and adenine phosphoribosyltransferase, in tk-, hgprt- or aprt- cells, respectively. Anti-metabolite resistance can also be used as the basis of selection for dhfr, which confers resistance to metrotexate; gpt that confers resistance to mycophenolic acid; neo that confers resistance to aminoglycoside G418; also that confers resistance to chlorosulfuron; and Hygro, which confers resistance to hygromycin. Additional selectable genes that can be used include trpB, which allows cells to use indole instead of tryptophan, or hisD, which allows cells to use histinol in place of histidine. Markers that give a visual indication for the identification of the transformant include anthocyanins, beta-glucuronidase and its substrate, GUS, and luciferase and its substrate, luciferin.
The polypeptide conjugates of the present invention can be produced using a combination of automatic peptide synthesis and recombinant techniques. For example, a polypeptide conjugate of the present invention can contain a combination of modifications including deletion, substitution, insertion and modification by pegylation (or other portion, eg, polymer, fatty acyl chain, C-terminal amidation). Said polypeptide conjugate can be produced in stages. In the first step, an intermediate polypeptide conjugate containing the deletion, substitution, insertion modifications and any combination thereof can be produced by recombinant techniques as described. After an optional purification step as described herein, the intermediate polypeptide conjugate is subjected to pegylation (or subjected to another chemical modification, for example acylation, C-terminal amidation) by chemical modification with a reagent suitable pegylation (for example from NeKtar Transforming Therapeutics, San Carlos, California), to produce the desired polypeptide conjugate derivative. The person skilled in the art will appreciate that the above described method can be generalized to be applied to a polypeptide conjugate containing a combination of selected modifications of deletion, substitution, insertion, derivation, and other very modifying means. known and contemplated by the present invention.
C-terminal amidation can be done using a C-terminally extended precursor of the amino acid glycine, synthesized for example in yeast (for example Pichia) as an alpha factor fusion protein that will be secreted into the culture medium. After purification, the C-terminal glycine of the precursor of the polypeptide conjugate will be converted to amide by enzymatic amidation, for example with peptidylglycine alpha-amidating monooxygenase (PAM); see for example Cooper et al., Biochem. Biophys. Acta, 1014: 247-258 (1989); see also US Pat. UU 6319685, which is incorporated herein by reference, which teaches methods for enzymatic amidation that include a rat alpha-amidating enzyme, sufficiently pure in alpha-amidating enzyme to exhibit a specific activity of at least about 25 mU per mg of protein, and substantially free of proteolytic impurities to be suitable for use with substrates purified from natural sources or produced by recombinant DNA techniques.
Formulations: The description also provides pharmaceutical compositions comprising at least one of the polypeptide conjugates described herein, and a pharmaceutically acceptable carrier. The polypeptide conjugates may be present in the pharmaceutical composition in a therapeutically effective amount and may be present in an amount to provide a minimum level in the blood plasma for therapeutic efficacy. The pharmaceutical compositions that contain the polypeptide conjugates described herein may be provided for peripheral administration, such as parenteral (e.g., subcutaneous, intravenous, intramuscular), topical, nasal or oral administration. Pharmaceutically acceptable carriers and their formulation are described in standard formulation treaties, such as Martin's "Remington's Pharmaceutical Sciences"; and Wang et al, Journal of Parenteral Science and Technology, Technical Report No. 10, Suppl. 42: 2S (1988).
The polypeptide conjugates described herein may be provided in parenteral compositions for injection or infusion. For example, they can be suspended in water; in an inert oil such as for example a vegetable oil (for example sesame oil, peanut, olive, etc.); or another pharmaceutically acceptable vehicle. In one embodiment, the polypeptide conjugates are suspended in an aqueous vehicle, for example in an isotonic buffer at a pH of about 3.0 to 8.0, or about 3.0 to 5.0. The compositions can be sterilized by conventional sterilization techniques, or they can be sterilized by filtration. The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH buffering agents. Useful buffers include, for example, acetic acid buffers. A slow depot preparation can be used, so that therapeutically effective amounts of the preparation can be delivered to the bloodstream for many hours or days after subcutaneous injection, transdermal injection or other delivery method. The desired sotonicity can be achieved using sodium chloride or other pharmaceutically acceptable agents such as dextrose, boric acid, sodium tartrate, propylene glycol, polyols (such as mannitol and sorbitol), or other inorganic or organic solutes. Sodium chloride is particularly preferred for buffers containing sodium ions.
Polypeptide conjugates can also be formulated as pharmaceutically acceptable salts (e.g., acid addition salts) and / or complexes thereof. The pharmaceutically acceptable salts are harmless salts at the concentration at which they are administered. Pharmaceutically acceptable salts include acid addition salts such as those containing sulfate, hydrochloride, phosphate, sulfamate, acetate, citrate, lactate, tartrate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, cyclohexyl sulfamate and quinate. The pharmaceutically acceptable salts can be obtained from acids such as hydrochloric acid, sulfuric acid, phosphoric acid, sulfamic acid, acetic acid, citric acid, lactic acid, tartaric acid, malonic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-acid toluenesulfonic acid, cyclohexylsulfamic acid, and chemical acid. The salt forms of acetate, trifluoroacetate or hydrochloride have particular use herein. Said salts can be prepared, for example, by reacting the free acid or base forms of the product with one or more equivalents of the base or acid appropriate, in a solvent or medium in which the salt is insoluble, or in a solvent such as water which is then removed under vacuum or by means of freeze drying, or by means of ion exchange of an existing salt by another ion on a suitable ion exchange resin.
Vehicles or excipients may also be used to facilitate administration of the polypeptide conjugates. Examples of carriers and excipients include calcium carbonate, calcium phosphate, various sugars such as lactose, glucose or sucrose, or types of starch, cellulose derivatives, gelatin, vegetable oils, polyethylene glycols and physiologically compatible solvents.
If desired, the solutions of the above compositions can be thickened with a thickening agent such as methylcellulose. They can be prepared in emulsified form, either water in oil or oil in water. A wide variety of pharmaceutically acceptable emulsifying agents can be used which include for example acacia powder, a nonionic surfactant (such as a Tween), or an ionic surfactant (such as alkali polyether alcohol sulfates or sulphonates, for example a Triton).
The compositions can be prepared by mixing the ingredients following the generally accepted procedures. For example, the selected components can be simply mixed in a mixer or other standard device to produce a concentrated mixture, which can then be adjusted to the concentration and viscosity final adding water or thickening agents, and possibly a buffer to control the pH, or an additional solute to control the tonicity.
The therapeutically effective amount of the polypeptide conjugates described herein to treat the diseases described herein will normally be about 0.01 pg about 5 mg; about 0.1 pg to about 2.5 mg; about 1 pg to about 1 mg; about 1 pg to about 50 pg; or about 1 pg to about 25 pg. Alternatively, the therapeutically effective amount of the GLP-1 receptor agonist polypeptide conjugates may be from about 0.001 pg to about 100 pg, based on the weight of a subject of 70 kg, or from about 0.01 pg to about 50 pg , based on the weight of a subject of 70 kg. These therapeutically effective doses can be administered once a day, twice a day, three times a day, once a week, biweekly, or once a month, depending on the formulation. The exact dose to be administered is determined, for example, according to the formulation, such as an immediate-release formulation or a sustained-release formulation. For transdermal, nasal or oral dosage forms, the dose may be increased from about 5 times to about 10 times.
The conjugated polypeptides described herein, which have an anti-hyperglycemia property with improved weight loss property, and pharmaceutical compositions comprising the conjugated polypeptides, are useful for the treatment of many metabolic diseases and conditions such as those described in the present, wherein there is a benefit to control the level of glucose in the blood, to reduce, prevent, or treat hyperglycemia and / or to control weight loss or food intake, to prevent, treat or control the overweight or obesity. The treatment or prevention of hyperglycemia is central to treating or preventing prediabetes, abnormal glucose intolerance, insulin resistance and diabetes. Diabetes can be type 1 diabetes, type 2 diabetes or gestational diabetes. The conjugated polypeptides present are very useful when these conditions, for example type 2 diabetes or type 1 diabetes, are associated with overweight or obesity or tendency to overweight or obesity, for example caused by overfeeding, stress, drugs that increase the weight such as insulin, or a genetic abnormality, or other diseases and conditions described herein that would be known to a physician skilled in the art. The methods to treat diabetes with and without the presence of overweight or obesity or tendency to it, provide administration to a subject, typically a subject in need thereof, of a therapeutically effective amount of one or more of the conjugated polypeptides described herein to treat the subject. The present polypeptide conjugates also have use in conditions where hyperglycemia is an important factor, often in the absence of overt diabetes, and in addition where overweight or obesity are present. present or may occur, such as in steroid-induced diabetes, diabetes induced by treatment against human immunodeficiency virus (HIV), adult latent autoimmune diabetes (LADA), non-alcoholic steatohepatitis (NASH) and non-alcoholic fatty liver disease (NAFLD), development of diabetes in subjects with congenital or HIV-associated lipoatrophy, or "fat redistribution syndrome", and in the metabolic syndrome (syndrome X).
Obesity and its associated disorders, including overweight, are common and serious public health problems in the United States. UU and all over the world. Obesity of the upper body is the strongest known risk factor for type 2 diabetes mellitus and is a strong risk factor for cardiovascular disease. Obesity is a recognized risk factor for hypertension, atherosclerosis, congestive heart failure, stroke, gallbladder disease, osteoarthritis, sleep apnea, reproductive disorders such as polycystic ovary syndrome, breast cancer, prostate and colon, and increased incidence of complications of general anesthesia; see for example Kopelman, 2000, Nature 404: 635-43.
Obesity reduces the maximum duration of life and carries a serious risk of the comorbidities mentioned above, and also disorders such as infections, varicose veins, acanthosis nigricans, eczema, exercise intolerance, insulin resistance, hypercholesterolemia of hypertension, cholelithiasis, injury orthopedic and thromboembolic disease; see for example Rissanen et al, 1990, Br. Med. J., 301: 835-7. Obesity is also a risk factor for the group of conditions called insulin resistance syndrome, or "syndrome X" and metabolic syndrome. The global medical cost of obesity and its associated disorders is enormous.
It is believed that the pathogenesis of obesity is multifactorial. One problem is that in obese subjects, nutrient availability and energy expenditure do not balance until there is an excess of adipose tissue. The central nervous system (CNS) controls the energy balance and coordinates a variety of behavioral, autonomous and endocrine activities appropriate to the animal's metabolic status. The mechanisms or systems that control these activities are widely distributed through the forebrain (for example the hypothalamus), the cerebellum (for example the brainstem) and the spinal cord. Finally, the metabolic (ie, fuel availability) and cognitive (that is, the learned preferences) information of these systems is integrated, and the decision to adopt an appetite (food-seeking) and consummatory (ingestion) behavior is activated (procuración and start of the meal) or off (termination of the meal). It is thought that the hypothalamus is primarily responsible for integrating these signals and after issuing commands to the brainstem. The brainstem nuclei that control the elements of the consummatory motor control system (for example, the muscles responsible for chewing and swallowing). Therefore, these CNS nuclei are literally referred to as constituents of "the final common path" of ingestive behavior.
Neuroanatomical and pharmacological evidence supports that energy signals and nutritional homeostasis are integrated into the nuclei of the forebrain and that the motor control system resides in the brainstem nuclei, probably in regions surrounding the trigeminal motor nucleus. There is nsive reciprocal connection between the hypothalamus and the brainstem. A variety of anti-obesity therapies targeting the CNS (eg, small molecules and peptides) are predominantly focused on the forebrain substrates that reside in the hypothalamus and / or substrates of the cerebellum that reside in the brainstem.
Obesity remains a poorly treatable, chronic, essentially intractable metabolic disorder. Accordingly, there is a need for new therapies useful in reducing weight and / or maintaining weight in a subject. These therapies would produce a profound beneficial effect on the health of the subject.
Diabetes and cardiovascular disease: Diabetes mellitus is recognized as a chronic complex disease where 60% to 70% of all fatal cases among diabetic patients are the result of cardiovascular complications. Diabetes is not only considered a risk of equivalent coronary heart disease, but is also identified as an independent predictor of adverse events that include recurrent myocardial infarction, congestive heart failure and death subsequent to a cardiovascular incident. It would be expected that the adoption of a narrower and more aggressive glucose control treatment for cardiovascular risk factors will decrease the risk of complications of coronary heart disease and improve overall survival among diabetic patients. However, diabetic patients are two to three times more likely to experience acute myocardial infarction than non-diabetic patients, and diabetic patients live eight to thirteen years less than non-diabetic patients.
Understanding of the high-risk nature of diabetic patients with acute myocardial infarction: American College of Cardiology / American Heart Association ("ACC / AHA") clinical practice guidelines for the management of hospitalized patients with unstable angina or infarction of myocardium without ST elevation (collectively referred to as "ACS") recently recognized that hospitalized diabetic patients are a special population that requires aggressive management of hyperglycemia. Specifically, the guidelines state that glucose reduction therapy for hospitalized diabetics / ACS patients should aim to achieve a prepandial glucose of less than 10 mg / dL, a maximum daily goal of 180 mg / dL, and a hemoglobin A1c after discharge. less than 7% In a national sample of elderly ACS patients, it was shown that a 30-day mortality increase in diabetic patients corresponds to patients who have higher glucose values after admission to the hospital; see "Diabetic Coronan / Artery Disease &Intervention", Coronary Therapeutics 2002, Oak Brook, IL, September 20, 2002. There is growing evidence that after admission to the hospital, sustained hyperglycemia, rather than transient elevated glucose, is related to serious adverse events. Although the ideal metric for hyperglycemia and vascular risk in patients is not known, it seems that the average glucose value during hospitalization is more predictive of mortality. In a separate study of ACS patients from more than 40 hospitals in the United States, it was found that persistent hyperglycemia, compared to randomized glucose values after admission to the hospital, was more predictive of mortality in the hospital; see "Acute Coronan / Syndrome Summit: A State of the Art Approach," Kansas City, MO, September 21, 2002. Compared to glucose values after admission, a logistic regression model of glucose control over the entire Hospitalization was more predictive of mortality. There was an almost double increased risk of mortality during hospitalization for each 10 mg / dl increase in glucose over 120 mg / dl. In a smaller cohort of consecutive diabetic / ACS patients, there was a graded increase in one-year mortality with increasing glucose levels after admission to the hospital. In the hospital setting, ACC / AHA guidelines suggest the initiation of aggressive insulin therapy to achieve lower blood glucose during hospitalization.
Lipid regulation diseases: As is known, lipodystrophy is characterized by abnormal or degenerative conditions of the adipose tissue of the body. Dyslipidemia is an interruption in the normal lipid component in the blood. It is believed that prolonged elevation of insulin levels can produce dyslipidemia. Hyperlipidemia is the presence of high or abnormal levels of lipids and / or lipoproteins in the blood. Hypothalamic amenorrhea is a condition in which menstruation stops for several months due to a problem involving the hypothalamus. It has been found that leptin replacement therapy in women with hypothalamic amenorrhea improves the axes of reproductive hormones, thyroid and growth, and markers of bone formation, without causing adverse effects; see, for example, Oral et al., N Engl J Med. 2004, 351: 959-962, 987-997. Fatty liver disease, for example, nonalcoholic fatty liver disease (NAFLD) refers to a broad spectrum of liver diseases ranging from simple fatty liver (steatosis) to nonalcoholic steatohepatitis (NASH) to cirrhosis (advanced irreversible scarring). of the liver). All stages of NAFLD have in common the accumulation of fat (fatty infiltration) in liver cells (hepatocytes). It is believed that leptin is one of the key regulators for the inflammation and progression of fibrosis in several chronic liver diseases that include NASH; see for example Ikejima et al., Hepatology Res. 33: 151-154.
Additionally, without wishing to be bound by any theory, it is believed that the relative insulin deficiency in type 2 diabetes, the glucose toxicity and the increase in the liver free fatty acid load by high delivery from the intraabdominal adipose tissue through of the portal vein, are implicated as possible causes of fatty liver disorders. In fact, it has been hypothesized that eating behavior is the key factor that manages the metabolic syndrome of obesity with its many corollaries, which include NASH. Therefore, it has already been shown that treatments aimed at decreasing food intake and increasing the number of small meals in type 2 diabetes can efficiently treat and prevent NASH. Drugs that promote insulin secretion and weight loss, and that delay gastric emptying, are also effective in improving glucose tolerance and thus improving fatty liver with its concomitant hyperinsulinemia. Thus, the use of exendins, exendin analog agonists, exendin-derived agonists, particularly exendin 4, may be very suitable as a treatment modality for this condition. Accordingly, the polypeptide conjugates described herein may be useful in the treatment of fatty liver disorders.
Metabolic syndrome X: Metabolic syndrome X is characterized by insulin resistance, dyslipidemia, hypertension and visceral distribution of adipose tissue, and has a pivotal role in the pathophysiology of type 2 diabetes. It has also been found to strongly correlate with NASH, fibrosis and cirrhosis of the liver. Accordingly, the polypeptide conjugates described herein may be useful in the treatment of metabolic syndrome X.
Steroid-induced diabetes: It is well known that glucocorticoids affect the metabolism of carbohydrates. In response to the administration of exogenous glucocorticoid, an increase in hepatic glucose production and reduction of insulin secretion and insulin-stimulated glucose uptake in peripheral tissues is observed. In addition, glucocorticoid treatment alters the proportion of proinsulin (P1) / immunoreactive insulin (IRI), as is known. Typical characteristics of glucocorticoid-induced hyperglycemia in subjects without diabetes include minimal fasting blood glucose elevation, exaggerated postprandial hyperglycemia, exogenous insensitivity to insulin, and lack of response to metformin or sulphonylurea therapy. Accordingly, the polypeptide conjugates described herein that include a biologically active amylin, exendin or davalantide peptide component (hormone domain), or a fragment or analogue thereof, may be useful in the treatment of induced diabetes by steroid.
Diabetes induced by treatment against the human immunodeficiency virus (HIV): Briefly after the introduction of protease inhibitors (PI) of the human immunodeficiency virus (HIV) -1 to routine clinical use, reports began to appear linking the use of PT with the development of hypergiucemia. Although approximately 1% to 6% of subjects infected with HIV who are treated with PI develop diabetes mellitus, a considerably larger proportion will develop insulin resistance and impaired glucose tolerance. Accordingly, polypeptide conjugates described herein that include a biologically active peptide component (hormone domain) of amylin, exendin or davalantide, or a fragment or analogue thereof, may be useful in the treatment of induced diabetes for treatment against HIV.
Adult latent autoimmune diabetes (LADA): It is thought that progressive autoimmune diabetes, also known as latent autoimmune diabetes of the adult (LADA), is present in approximately 10% of patients diagnosed with type 2 diabetes. LADA patients have circulating antibodies to an islet cell cytoplasmic antigen or, more frequently, glutamic acid decarboxylase. These subjects exhibit clinical features characteristic of both type 1 and type 2 diabetes. Although insulin secretion is better preserved in the slowly progressing form than in the rapidly advancing form of autoimmune diabetes, insulin secretion tends to deteriorate with the time in the LADA subjects. Accordingly, polypeptide conjugates described herein that include a biologically active amylin, exendin or davalantide peptide component (hormone domain), or a fragment or analogue thereof, may be useful in the treatment of LADA.
The polypeptide conjugates described herein and the pharmaceutical compositions comprising the polypeptide conjugates are useful for the treatment of insulin resistance and for stimulating the release of insulin. Polypeptide conjugates are particularly useful when such conditions are additionally associated with overweight or obesity, or a tendency to be overweight or obese. Methods for the treatment of insulin resistance provide administration to a subject in need thereof of a therapeutically effective amount of a polypeptide conjugate described herein to treat insulin resistance in the subject. Treatment methods for stimulating insulin release provide for administration to a subject in need thereof of a therapeutically effective amount of a polypeptide conjugate described herein, to stimulate the release of insulin in the subject.
The polypeptide conjugates described herein and the pharmaceutical compositions comprising the polypeptide conjugates are useful for the treatment of postprandial hyperglycemia. Polypeptide conjugates are particularly useful when such conditions are additionally associated with overweight and obesity, or a tendency to be overweight or obese. Methods of treating postprandial hyperglycemia provide for administration to a subject in need thereof of a therapeutically effective amount of a polypeptide conjugate described herein, to treat postprandial hyperglycemia in the subject.
The polypeptide conjugates described herein and the pharmaceutical compositions comprising the polypeptide conjugates are useful for reducing blood glucose levels and reducing HbA1c levels. Polypeptide conjugates are particularly useful when such conditions are additionally associated with overweight or obesity or with a tendency to overweight or obesity. Polypeptide conjugates are particularly useful when such conditions are additionally associated with overweight or obesity, or a tendency to overweight or obesity. Methods for reducing blood glucose levels provide for administration to a subject in need thereof of a therapeutically effective amount of a polypeptide conjugate described herein, to reduce the blood glucose levels of the subject. In one embodiment, blood glucose levels may be fasting blood glucose levels. Methods for reducing HbA1c levels provide for administration to a subject in need thereof of a therapeutically effective amount of a polypeptide conjugate described herein, to reduce HbA1c levels in the subject. HbA1c levels are usually a long-term measure of the patient's blood glucose levels.
Also provided are methods for the treatment of diabetes, for example type 1, type 2, or gestational diabetes, which comprise administering a sufficient polypeptide conjugate to obtain an average or minimum circulating level of a polypeptide conjugate in the blood plasma. of at least about 50 pg / ml for a period of at least about 12 hours, at least about 1 day, at least about 2 days, at least about 3 days, at least about 1 week, so less approximately 2 weeks, at least approximately 3 weeks, at least about 1 month, at least about 3 months, or at least about 6 months. In one embodiment, the methods comprise administering a polypeptide conjugate sufficient to obtain a minimum or average circulating blood plasma concentration of at least about 25 pg / ml, at least about 50 pg / ml, at least about 65 pg / ml, at least about 75 pg / ml, at least about 100 pg / ml, at least about 150 pg / ml, at least about 170 pg / ml, at least about 175 pg / ml, at least about 200 pg / ml, at least about 225 pg / ml, at least about 250 pg / ml, at least about 350 pg / ml, at least about 400 pg / ml, at least about 450 pg / ml, at least about 500 pg / ml, at least about 550 pg / ml or at least about 600 pg / ml of the polypeptide conjugate. In other embodiments, the average or minimum concentration of the polypeptide conjugate is between at least about 170 pg / ml and 600 pg / ml, or between at least about 170 pg / ml and 350 pg / ml. In other embodiments, the average or minimum plasma concentration of the polypeptide conjugate is greater than 40 pmol / liter, greater than 50 pmol / liter, greater than 60 pmol / liter, greater than 70 pmol / liter, greater than 80 pmol / liter, greater than 90 pmol / liter, greater than 100 pmol / liter, greater than 110 pmol / liter, greater than 120 pmol / liter, greater than 130 pmol / liter, greater than 140 pmol / liter, or greater than 150 pmol / liter. In additional embodiments, the average or minimum plasma concentration of the polypeptide conjugate is greater than 40 pmol / liter but less than 150 pmol / liter, or greater than 40 pmol / liter but less than 80 pmol / liter. In one embodiment, the polypeptide conjugate is compound 1A or compound 2A, or compound 1A or a derivative thereof. In other embodiments, the concentration of the polypeptide conjugate is the concentration of a polypeptide conjugate that results in a biological or therapeutic effect, for example reduction of fasting glucose, reduction of postprandial glucose excursion, reduction of HbA1c, etc. , equivalent to that observed with a given concentration of exendin 4, compound 4A, davalantide, or a combination of exendin plus davalantide. In additional modalities, the subject is in need or desire to reduce his body weight. In a further embodiment, the subject is also in need of controlling his weight / food intake, for example in need of reducing body weight, reducing appetite, increasing satiety, reducing food intake, delaying gastric emptying, reducing triglycerides, improving body composition, or any combination thereof, and optionally also with lower incidence and / or severity of nausea.
Additional modalities provide methods of reducing HbA1c, the average daily concentration of glucose in the blood, the fasting blood glucose and / or the postprandial blood glucose, for example, administering a subject in need of reducing HbA1c, the average daily glucose in the blood , or fasting glucose, a sufficient amount of polypeptide conjugate to obtain an average circulating level Minimum in the blood plasma of an exendin, a polypeptide conjugate, of at least about 50 pg / ml for a period of at least about 12 hours, at least about 1 day, at least about 2 days, at least about 3 days, at least about 1 week, at least about 2 weeks, at least about 3 weeks, at least about 1 month, at least about 3 months, or at least approximately 6 months. In one embodiment, the methods comprise administering a sufficient polypeptide conjugate to obtain a minimum or average circulating blood plasma concentration of at least about 25 pg / ml, at least about 65 pg / ml, at least about 75 pg / ml, at least about 100 pg / ml, at least about 150 pg / ml, at least about 170 pg / ml, at least about 175 pg / ml, at least about 200 pg / ml, at least about 225 pg / ml, at least about 250 pg / ml, at least about 350 pg / ml, at least about 400 pg / ml, at least about 450 pg / ml, at least about 500 pg / ml, at least about 550 pg / ml or at least about 600 pg / ml of the polypeptide conjugate. In other embodiments, the average or minimum concentration of the polypeptide conjugate is between at least about 170 pg / ml and 600 pg / ml, or between at least about 170 pg / ml and 350 pg / ml. In other embodiments, the average or minimum plasma concentration of the polypeptide conjugate is greater than 40 pmol / liter, greater than 50 pmol / liter, greater than 60 pmol / liter, greater than 70 pmol / liter, greater than 80 pmol / liter, greater than 90 pmol / liter, greater than 100 pmol / liter, greater than 110 pmol / liter, greater than 120 pmol / liter, greater than 130 pmol / liter, greater than 140 pmol / liter, or greater than 150 pmol / liter. In additional embodiments, the average or minimum plasma concentration of the polypeptide conjugate is greater than 40 pmol / liter but less than 150 pmol / liter, or greater than 40 pmol / liter but less than 80 pmol / liter. In a modality, the polypeptide conjugate is compound 1A or compound 2A, or compound 1A or a derivative thereof. In other embodiments, the concentration of the polypeptide conjugate is the concentration of a polypeptide conjugate that results in a biological or therapeutic effect, for example reduction of HbA1c, equivalent to that observed with a given concentration of exendin 4, compound 4A, davalantide, or a combination of exendin plus davalantide. In one embodiment, average or minimum blood circulating concentrations are obtained over a period of about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, or about 7 days. In a further embodiment, average or minimum plasma concentrations are obtained over a period of approximately 1 week, approximately 2 weeks. weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, about 11 weeks, about 12 weeks, about 13 weeks, about 14 weeks, approximately 15 weeks or approximately 16 weeks. In a further embodiment, the average or minimum plasma concentrations are obtained during a period of approximately 5 months, approximately 6 months, approximately 7 months, approximately 8 months, approximately 9 months, approximately 10 months, approximately 11 months, or approximately 12 months. To determine the circulating blood concentrations of exendin or exendin agonist or davalintide, any method can be used. In additional modalities, the subject has the need or desire to reduce his body weight. In a further embodiment, the subject also has the need to control his weight / food intake, for example the need to reduce body weight, reduce appetite, increase satiety, reduce food intake, slow gastric emptying, reduce triglycerides, improving body composition, or any combination thereof, and optionally also with lower incidence and / or severity of nausea.
Additionally, a method is provided to reduce the increase of the postprandial glucose concentration in the blood, compared to the preprandial glucose concentration in the blood, in such a way that the difference between the concentration of glucose in the blood before and after after a meal This results in a decrease in the variation of blood glucose concentrations during the day, determined for example by means of monitoring 7 blood glucose points as described herein. This method comprises administering an amount of a polypeptide conjugate, sufficient to obtain an average or minimum circulating level of a polypeptide conjugate in the blood plasma of at least about 50 pg / ml for a period of at least about 12 hours, at least about 1 day, at least about 2 days, at least about 3 days, at least about 1 week, at least about 2 weeks, at least about 3 weeks, at least about 1 month, at least about 3 months, or at least about 6 months. In one embodiment, the methods comprise administering a sufficient polypeptide conjugate to obtain an average or minimum circulating plasma concentration in the blood plasma of at least about 25 pg / ml, at least about 65 pg / ml, at least about 75 pg / ml, at least about 100 pg / ml, at least about 150 pg / ml, at least about 170 pg / ml, at least about 175 pg / ml, at least about 200 pg / ml , at least about 225 pg / ml, at least about 250 pg / ml, at least about 350 pg / ml, at least about 400 pg / ml, at least about 450 pg / ml, at least about 500 pg / ml, at least about 550 pg / ml or at least about 600 pg / ml of the polypeptide conjugate. In other embodiments, the average or minimum concentration of the polypeptide conjugate is between at least about 170 pg / ml and 600 pg / ml, or between at least about 170 pg / ml and 350 pg / ml. In other embodiments, the average or minimum plasma concentration of the polypeptide conjugate is greater than 40 pmol / liter, greater than 50 pmol / liter, greater than 60 pmol / liter, greater than 70 pmol / liter, greater than 80 pmol / liter , greater than 90 pmol / liter, greater than 100 pmol / liter, greater than 110 pmol / liter, greater than 120 pmol / liter, greater than 130 pmol / liter, greater than 140 pmol / liter, or greater than 150 pmol / liter. In more embodiments, the average or minimum plasma concentration of the polypeptide conjugate is greater than 40 pmol / liter but less than 150 pmol / liter, or greater than 40 pmol / liter but less than 80 pmol / liter. In one embodiment, the polypeptide conjugate is compound 1A or compound 2A, or compound 1A or a derivative thereof. In other embodiments, the concentration of the polypeptide conjugate is the concentration of a polypeptide conjugate that results in a biological or therapeutic effect, for example, reduction of postprandial glucose excursions in the blood, average daily blood glucose, etc. , equivalent to that observed with a given concentration of exendin 4, compound 4A, davalintide, or a combination of exendin plus davalintide.
In one embodiment, average or minimum blood circulating concentrations are obtained over a period of about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, or about 7 days. In a further embodiment, average or minimum plasma concentrations are obtained over a period of about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, approximately 10 weeks, approximately 11 weeks, approximately 12 weeks, approximately 13 weeks, approximately 14 weeks, approximately 15 weeks or approximately 16 weeks. In a further embodiment, average or minimum plasma concentrations are obtained over a period of about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, or about 12 months. To determine the circulating blood concentrations of the exendin or exendin agonist or davalintide, any method can be used. In additional modalities, the subject has the need or desire to reduce his body weight. In a further embodiment, the subject also has the need to control his weight / food intake, for example the need to reduce body weight, reduce appetite, increase satiety, reduce food intake, delay gastric emptying, reduce triglycerides, improving body composition, or any combination thereof, and optionally with lower incidence and / or severity of nausea.
The polypeptide conjugates described herein and the pharmaceutical compositions comprising the polypeptide conjugates are useful for reducing gastric motility and delaying gastric emptying. Polypeptide conjugates are particularly useful when such conditions are additionally associated with overweight or obesity or a tendency to overweight or obesity. Methods for reducing gastric motility provide administration to a subject in need thereof of a therapeutically effective amount of a polypeptide conjugate described herein to reduce gastric motility in the subject. Methods for delaying gastric emptying provide for administration to a subject in need thereof of a therapeutically effective amount of a polypeptide conjugate described herein, to delay gastric emptying in the subject.
The polypeptide conjugates described herein and the pharmaceutical compositions comprising the polypeptide conjugates are useful for reducing food intake, reducing appetite, increasing satiety, and reducing weight. Polypeptide conjugates are particularly useful when such conditions are additionally associated with hyperglycemia, for example diabetes or a tendency to hyperglycemia Methods for reducing food intake provide for administration to a subject in need thereof of a therapeutically effective amount of a polypeptide conjugate described herein, to reduce food intake in the subject. Methods for reducing appetite or increasing satiety provide for administration to a subject in need thereof of a therapeutically effective amount of a polypeptide conjugate described herein, to reduce appetite in the subject. Methods for reducing weight provide administration to a subject in need thereof of a therapeutically effective amount of a polypeptide conjugate described herein to reduce weight in the subject. In the methods described herein, the subject may be in need of reducing food intake, reducing appetite, or reducing weight. In other methods described herein, the subject may be willing to have a reduced feed intake, or have reduced appetite, or have reduced weight. The subject can be of any weight and can be obese or overweight.
The polypeptide conjugates described herein and the pharmaceutical compositions comprising the polypeptide conjugates are useful for the treatment of overweight and obesity. Polypeptide conjugates are particularly useful when such conditions are additionally associated with hyperglycemia, for example, diabetes, or a tendency to hyperglycemia. The methods for the treatment of overweight provide the administration to a subject in need thereof of a Therapeutically effective amount of a polypeptide conjugate described herein, to treat overweight in the subject. Methods for the treatment of obesity provide for administration to a subject in need thereof of a therapeutically effective amount of a polypeptide conjugate described herein, to treat obesity in the subject.
The polypeptide conjugates described herein and the pharmaceutical compositions comprising the polypeptide conjugates are useful for improving the body composition, for example with an improved ratio of lean muscle to body fat, usually in conjunction with a weight reduction. Polypeptide conjugates are particularly useful when such conditions are additionally associated with hyperglycemia, for example, diabetes, or a tendency to hyperglycemia. Methods for improving body composition provide for administration to a subject in need thereof of a therapeutically effective amount of a polypeptide conjugate described herein, to improve the subject's body composition and optionally reduce its weight.
In one embodiment, the present application provides methods for reducing weight in a subject with the desire or need for the same, wherein the method comprises administering an amount of a polypeptide conjugate effective to cause a weight reduction in the subject. In another embodiment, the method comprises chronic or sustained administration of a polypeptide conjugate effective to cause weight reduction in the subject. In another embodiment, the reduction in weight is due to a reduction in body fat or adipose tissue without a corresponding reduction in lean or muscular body mass. In another embodiment, the reduction in body weight due to the loss of body fat is greater than the reduction in weight due to the loss of lean or muscular body mass. In one modality, the reduction of body fat compared to lean tissue or muscle is based on absolute weight, while in another modality it is based on the percentage of weight lost. In one modality, the loss of visceral fat is greater than the loss of non-visceral fat. In another modality, the loss of non-visceral fat is greater than the loss of visceral fat. In yet another embodiment, the application provides methods for altering the body composition, for example reducing the proportion of fatty tissue to lean, reducing the percentage of body fat, or increasing the percentage of lean tissue in an individual.
As used herein, "weight reduction" refers to a decrease in the body weight of a subject. In one embodiment, the decrease in body weight is the result of a preferential decrease in the subject's body fat. In another modality, the loss of visceral fat is greater than the loss of non-visceral fat. In another modality, the loss of non-visceral fat is greater than the loss of visceral fat. Although the invention does not depend on any particular reduction in subject weight, the methods described herein, in various embodiments, will reduce the subject's weight by at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 10%, at least about 15, at least about 20%, at least about 30 %, at least about 40%, at least about 50%, at least about 60%, or at least about 70%, compared to the subject's body weight before starting the methods described herein. In various embodiments, the weight reduction occurs over a period of about 1 week, about 2 weeks, about 3 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, approximately 8 months, approximately 9 months, approximately 10 months, approximately 11 months, approximately 1 year, or more. In other embodiments, the subject may lose approximately 2.3 kg, approximately 2.7 kg, approximately 3.2 kg, approximately 3.6 kg, approximately 4.1 kg, approximately 4.5 kg, approximately 6.8 kg, approximately 9.1 kg, approximately 11.3 kg, approximately 13.6 kg, approximately 15.9 kg, approximately 18.1 kg, approximately 20.4 kg, approximately 22.7 kg, approximately 45.4 kg, approximately 56.7 kg, approximately 68 kg, approximately 79.4 kg, approximately 90.7 kg, or more. A weight reduction can be measured using any reproducible measurement means. In one embodiment, weight reduction can be measured by calculating a subject's body mass index and comparing that subject's BMI over a period. The body mass index can be calculated using any available method, for example using a nomogram or similar device.
The polypeptide conjugates described herein and the pharmaceutical compositions comprising the polypeptide conjugates are useful for the treatment of dyslipidemia. Polypeptide conjugates are particularly useful when such a condition is additionally associated with hyperglycemia, for example, diabetes, or a tendency to hyperglycemia. Methods for the treatment of dyslipidemia provide for administration to a subject in need thereof of a therapeutically effective amount of a polypeptide conjugate described herein to treat dyslipidemia in the subject. Methods for treating dyslipidemia provide for administration to a subject in need thereof of a therapeutically effective amount of a polypeptide conjugate described herein, to treat dyslipidemia in the subject.
The polypeptide conjugates described herein and the pharmaceutical compositions comprising the polypeptide conjugates are useful for the treatment of hypertriglyceridemia. Polypeptide conjugates are particularly useful when such a condition is additionally associated with hyperglycemia, for example, diabetes, or a tendency to hyperglycemia. Methods for the treatment of hypertriglyceridemia provide for administration to a subject in need thereof of a therapeutically effective amount of a polypeptide conjugate described herein to treat hypertriglyceridemia in the subject. Methods for the treatment of hypertriglyceridemia provide for administration to a subject in need thereof of a therapeutically effective amount of a polypeptide conjugate described herein, to treat hypertriglyceridemia in the subject.
The polypeptide conjugates described herein and the pharmaceutical compositions comprising the polypeptide conjugates are useful for the treatment of steroid-induced diabetes, diabetes induced by treatment against human immunodeficiency virus (HIV), latent autoimmune diabetes of the adult (LADA), nonalcoholic steatohepatitis (NASH) and nonalcoholic fatty liver disease (NAFLD), development of diabetes in subjects with congenital or associated lipoatrophy with HIV, or "fat redistribution syndrome", and / or metabolic syndrome ( syndrome X). Polypeptide conjugates are particularly useful when such conditions are additionally associated with hyperglycemia, for example, diabetes, or a tendency to hyperglycemia, or overweight or obesity, or a tendency to overweight or obesity. Methods for the treatment of steroid-induced diabetes, diabetes induced by treatment against human immunodeficiency virus (HIV), adult latent autoimmune diabetes (LADA), nonalcoholic steatohepatitis (NASH) and nonalcoholic fatty liver disease ( NAFLD), development of diabetes in subjects with lipoatrophy congenital or associated with HIV, or "fat redistribution syndrome", and / or metabolic syndrome (syndrome X), provide for administration to a subject in need thereof of a therapeutically effective amount of a polypeptide conjugate described herein for treat the disease / condition in the subject. Methods for the treatment of steroid-induced diabetes, diabetes induced by treatment against human immunodeficiency virus (HIV), adult latent autoimmune diabetes (LADA), nonalcoholic steatohepatitis (NASH) and nonalcoholic fatty liver disease ( NAFLD), development of diabetes in subjects with congenital or associated lipoatrophy with HIV, or "fat redistribution syndrome", and / or metabolic syndrome (syndrome X), provide administration to a subject in need thereof of a therapeutically effective amount of a polypeptide conjugate described herein to treat the disease / condition in the subject.
In the methods described herein, the subject may be in need of the aforementioned therapeutic effect and / or may be willing to have the aforementioned therapeutic effect. The subject can be of any weight and may be overweight or obese.
The methods of the present contemplate chronic or sustained administration of an effective amount of a polypeptide conjugate to a subject, to effect the desired results described herein.
The described methods can be used in any subject individual in need of such methods or in individual subjects for whom the practice of methods is desired. These individuals can be any mammal that includes, without limitation, humans, dogs, horses, cows, pigs and other commercially valuable animals or companion animals.
In some embodiments, the polypeptide conjugate is given by chronic administration. As used herein, "chronic administration" refers to the administration of the agent in a continuous mode, as opposed to an acute mode, in order to maintain the plasma concentration necessary to obtain the desired therapeutic effect (activity) over a prolonged period. . In one aspect, "chronic administration" refers to the administration of the polypeptide conjugate in a continuous mode, in order to maintain a plasma concentration in the therapeutically effective or desired amount, or above it. In one embodiment, such chronic administration maintains an average plasma concentration of the polypeptide conjugate of at least about 25 pg / ml, at least about 50 pg / ml, at least about 65 pg / ml, at least about 75 pg. / ml, at least about 85 pg / ml, at least about 100 pg / ml, at least about 150 pg / ml, at least about 170 pg / ml, at least about 175 pg / ml, so less about 200 pg / ml, at least about 225 pg / ml, at least about 250 pg / ml, at least about 300 pg / ml, at least about 350 pg / ml, at least about 400 pg / ml, at least about 450 pg / ml, at least about 500 pg / ml, at least about 550 pg / ml or at least about 600 pg / ml , for a prolonged period. In other embodiments, the average concentration of the polypeptide conjugate is between at least about 170 pg / ml and 600 pg / ml, or between at least about 170 pg / ml and 350 pg / ml. In other embodiments, the average plasma concentration of the polypeptide conjugate is greater than 40 pmol / liter, greater than 50 pmol / liter, greater than 60 pmol / liter, greater than 70 pmol / liter, greater than 80 pmol / liter, greater than 90 pmol / liter, greater than 100 pmol / liter, greater than 110 pmol / liter, greater than 120 pmol / liter, greater than 130 pmol / liter, greater than 140 pmol / liter, or greater than 150 pmol / liter. In additional embodiments, the average plasma concentration of the polypeptide conjugate is greater than 40 pmol / liter but less than 150 pmol / liter, or greater than 40 pmol / liter but less than 80 pmol / liter. In one embodiment, the polypeptide conjugate is compound 1A or compound 2A, or in other embodiments is compound 1A or its derivatives. In other embodiments, the concentration of the polypeptide conjugate is the concentration of polypeptide conjugate that results in a biological or therapeutic effect, e.g., weight reduction, glucose reduction, alteration of body composition, etc., ealent to that observed with a given concentration of exendin 4, compound 4A, davalintide, or a combination of exendin plus davalintide.
In another embodiment, the chronic administration maintains the plasma concentration, average or minimum, of the polypeptide conjugate for a period of at least about 12 hours or at least about 1 day, at least about 2 days, at least about 3 hours. days, at least about 4 days, at least about 5 days, at least about 6 days, or at least about 7 days. In another embodiment, chronic administration maintains the plasma concentration of the polypeptide conjugate for at least 1 week, at least about 2 weeks, at least about 3 weeks, or at least about 4 weeks, or at least about 1 month, at least about 2 months, or at least about 3 months. In other embodiments, the polypeptide conjugate is administered continuously. As used herein, the "continuous manner" refers to the introduction of the polypeptide conjugate to the body, for example into the circulation, and not the means of administration. Thus, chronic administration on a continuous basis may result from a continuous infusion, either intravenously or subcutaneously; of the use of a pump or dosing system, implanted or external, for the continuous or intermittent supply; or using an extended-release, slow-release, sustained-release or long-acting formulation, which is administered, for example, once a day, twice a week, weekly, twice a month, every month, a month and another no or every third month. It must be recognized that it is not necessary to reach immediately the average or minimum plasma level after the administration of the formulation, but it can take from hours to days or weeks. Once this level is reached, the average or minimum plasma concentration is maintained during the desired period to exert its therapeutic effect.
As used herein in the context of weight reduction or alteration of body composition, a "subject in need thereof" is a subject who is overweight or obese. As used herein in the context of weight reduction or alteration of body composition, a "willing" subject is a subject who wants to reduce their body weight or alter their body composition, for example by decreasing their proportion of fat to lean tissue. In one embodiment, the subject is an obese or overweight subject. In exemplary embodiments, an "overweight subject" refers to a subject with a body mass index (BMI) greater than 25, or a BMI between 25 and 30. However, it must be recognized that the meaning of overweight is not limited to individuals with a BMI greater than 25, but it refers to any subject in whom weight loss is desirable, or is indicated for medical or cosmetic reasons. Although "obesity" is generally defined as a body mass index greater than 30, for the purposes of this description, any subject who needs or wishes to reduce body weight is included in the scope of the term "obsessive". In one embodiment, subjects who are insulin resistant, glucose intolerant, or who have any form of diabetes mellitus (e.g., type 1, type 2, or gestational diabetes) can benefit from this method. In another modality, a Subject in need thereof is obese. However, it should be noted that the method described herein can be applied to subjects who do not have, and / or who have not been diagnosed with, impaired glucose tolerance, insulin resistance or diabetes mellitus.
As used herein in the context of the treatment of diabetes, reduction of HbA1c, control of postprandial blood glucose, reduction of fasting glucose and reduction of the general daily concentration of glucose in the blood, a subject in need thereof can include subjects with diabetes, impaired glucose tolerance, insulin resistance, or subjects unable to self-regulate their blood glucose.
Hbalc or A1c or glycated hemoglobin or glycohemoglobin, as commonly used, refers to glycosylated hemoglobin.
In one embodiment methods are provided for reducing body weight, reducing the proportion of fat to lean tissue, or reducing BMI, wherein the methods comprise chronically administering the polypeptide conjugate to a subject in need or desire thereof. In one embodiment, the weight loss attributed to the loss of fat or adipose tissue is greater than the loss of weight due to lean tissue. In another embodiment, the percentage of weight reduction due to loss of lean body mass is less than about 40%, less than about 30%, less than about 20%, less than about 10%, less than about 5%, less of about 2%, less than about 1%, or 0% of the total weight reduction. In one embodiment, the polypeptide conjugate is administered in an extended release, slow release, sustained release, or long-acting formulation. In one embodiment, the polypeptide conjugate is administered in a polymer-based sustained release formulation. Sustained-release formulations based on polymer of this type are described, for example, in U.S. Patent Application Ser. UU Serial No. 09 / 942,631, filed August 31, 2001 (now U.S. Patent No. 6,824,822) and related application Serial No. 11 / 312,371, filed December 21, 2005; the provisional application of EE. UU No. 60 / 419,388, filed October 17, 2002, and US patent applications. UU related, Nos. series 10 / 688,786 and 10 / 688,059, filed on October 17, 2003; the provisional application of EE. UU No. 60 / 757,258, filed January 9, 2006; the provisional application of EE. UU Serial No. 60 / 563,245, filed April 15, 2004, and the US patent application. UU related Serial No. 11 / 104,877, filed on April 13, 2005; and the US patent application. UU Serial No. 11 / 107,550, filed on April 15, 2005; all of which is incorporated herein by reference.
In any of the embodiments or methods described herein, circulating plasma concentrations of the polypeptide conjugate can be maintained at a given average plasma concentration or within about 10%, about 15%, about 20%, or about 25% of the average plasma concentration given. In other modalities, concentrations circulating plasma levels are maintained at the given average concentration or at approximately 98%, approximately 97%, approximately 96%, approximately 95%, approximately 90%, approximately 80%, approximately 70%, or approximately 60% of the given average concentration. Plasma concentrations of the polypeptide conjugate can be measured using any method available to the person skilled in the art.
In any of the embodiments or methods described herein, administration of the polypeptide conjugate is effective to support a minimum circulating plasma concentration of the polypeptide conjugate of at least about 50 pg / ml for at least about 12 hours, approximately 24 hours per day. hours or approximately 48 hours. In other embodiments, the methods comprise administering sufficient polypeptide conjugate to support a minimum circulating plasma concentration of at least about 25 pg / ml, at least about 65 pg / ml, at least about 75 pg / ml, per at least about 100 pg / ml, at least about 150 pg / ml, at least about 170 pg / ml, at least about 175 pg / ml, at least about 200 pg / ml, at least about 225 pg / ml, at least about 250 pg / ml, at least about 350 pg / ml, at least about 400 pg / ml, at least about 450 pg / ml, at least about 500 pg / ml, so less about 550 pg / ml or at least about 600 pg / ml of the polypeptide conjugate. In other embodiments, the minimum concentration of the polypeptide conjugate is between at least about 170 pg / ml and 600 pg / ml, or between at least about 170 pg / ml and 350 pg / ml. In other embodiments, the minimum plasma concentration of the polypeptide conjugate is greater than 40 pmol / liter, greater than 50 pmol / liter, greater than 60 pmol / liter, greater than 70 pmol / liter, greater than 80 pmol / liter, higher of 90 pmol / liter, greater than 100 pmol / liter, greater than 110 pmol / liter, greater than 120 pmol / liter, greater than 130 pmol / liter, greater than 140 pmol / liter, or greater than 150 pmol / liter. In more embodiments, the minimum plasma concentration of the polypeptide conjugate is greater than 40 pmol / liter but less than 150 pmol / liter, or greater than 40 pmol / liter but less than 80 pmol / liter. In one embodiment, the polypeptide conjugate is compound 1A or compound 2A, or compound 1A or a derivative thereof. In other embodiments, the concentration of the polypeptide conjugate is the concentration of a polypeptide conjugate resulting in a biological or therapeutic effect, for example, weight reduction, glucose reduction, alteration of body composition, etc., equivalent to that observed with a given concentration of exendin 4, compound 4A, davalintide, or a combination of exendin plus davalintide. In some embodiments, the minimum concentration of the polypeptide conjugate is sustained for a period of at least about 2 days, at least about 3 days, at least about 4 days, so less about 5 days, at least about 6 days, or at least about 7 days. In various embodiments, the minimum circulating plasma concentrations are sustained for at least about 2 weeks, at least about 3 weeks, at least about 4 weeks, at least about 5 weeks, at least about 6 weeks, at least approximately 7 weeks, at least about 8 weeks, at least about 9 weeks, at least about 10 weeks, at least about 11 weeks, at least about 12 weeks, at least about 13 weeks, at least about 14 weeks, at least approximately 15 weeks or at least approximately 16 weeks. In additional embodiments, the minimum circulating plasma levels are sustained for at least about 5 months, at least about 6 months, at least about 7 months, at least about 8 months, at least about 9 months, at least approximately 10 months, at least approximately 11 months or at least approximately 12 months. Plasma concentrations of the polypeptide conjugate can be measured using any method available to the person skilled in the art.
In any of the embodiments or methods described herein, administration of the polypeptide conjugate is effective to maintain average plasma concentrations of the polypeptide conjugate of at least about 50 pg / ml for at least about 12 hours, at least about 24 hours or at least approximately 48 hours. In other embodiments, the methods comprise administering a polypeptide conjugate sufficient to sustain an average circulating plasma concentration of at least about 25 pg / ml, at least about 65 pg / ml, at least about 75 pg / ml, at least about 100 pg / ml, at least about 150 pg / ml, at least about 170 pg / ml, at least about 175 pg / ml, at least about 200 pg / ml, at least about 225 pg / ml, at least about 250 pg / ml, at least about 350 pg / ml, at least about 400 pg / ml, at least about 450 pg / ml, at least about 500 pg / ml, per at least about 550 pg / ml or at least about 600 pg / ml. In other embodiments, the average concentration of the polypeptide conjugate is between at least about 170 pg / ml and 600 pg / ml, or between at least about 170 pg / ml and 350 pg / ml. In other embodiments, the average plasma concentration of the polypeptide conjugate is greater than 40 pmol / liter, greater than 50 pmol / liter, greater than 60 pmol / liter, greater than 70 pmol / liter, greater than 80 pmol / liter, higher of 90 pmol / liter, greater than 100 pmol / liter, greater than 110 pmol / liter, greater than 120 pmol / liter, greater than 130 pmol / liter, greater than 140 pmol / liter, or greater than 150 pmol / liter.
In additional embodiments, the average plasma concentration of the polypeptide conjugate is greater than 40 pmol / liter but less than 150 pmol / liter, or greater than 40 pmol / liter but less than 80 pmol / liter. In one embodiment, the polypeptide conjugate is compound 1A or compound 2A, or compound 1A or a derivative thereof. In other embodiments, the concentration of the polypeptide conjugate is the concentration of a polypeptide conjugate that results in a biological or therapeutic effect, for example, weight reduction, glucose reduction, alteration of body composition, etc. equivalent to that observed with a given concentration of exendin 4, compound 4A, davalintide, or a combination of exendin plus davalintide. In some embodiments, the average concentration of the polypeptide conjugate is sustained for a period of at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days. days, or at least approximately 7 days. In various embodiments, the average circulating plasma concentrations are sustained for at least about 2 weeks, at least about 3 weeks, at least about 4 weeks, at least about 5 weeks, at least about 6 weeks, at least about 7 weeks, at least about 8 weeks, at least about 9 weeks, at least about 10 weeks, at least about 1 week, at least about 12 weeks, at least about 13 weeks, at least about 14 weeks, at least about 15 weeks, or at least about 16 weeks. In additional embodiments, the average circulating plasma levels are sustained for at least about 5 months, at least about 6 months, at least about 7 months, at least about 8 months, at least about 9 months, at least approximately 10 months, at least approximately 11 months, or at least approximately 12 months. Plasma concentrations of the polypeptide conjugate can be measured using any method available to the person skilled in the art.
The polypeptide conjugate can be administered by any available method. In one embodiment, the polypeptide conjugate is administered subcutaneously. In another embodiment, the polypeptide conjugate is administered orally or by means of a pump or implant. In one embodiment the polypeptide conjugate is administered continuously. In another embodiment, the polypeptide conjugate is administered in a slow release, extended release, sustained release, or long-acting formulation. In any of the foregoing embodiments, the polypeptide conjugate can be administered once a day, every other day, three times a week, twice a week, once a week, twice a month, each month, one month yes and the other no, or every three months. Further, the total duration of administration of the polypeptide conjugate can be determined by the desired amount of weight reduction. Thus, the polypeptide conjugate can be administered according to the methods described herein for a period sufficient to achieve a given weight, BMI or target body composition, after which the administration can be terminated. Alternatively, after achieving the weight, BMI or target body composition, the dose of the polypeptide conjugate can be reduced to a sufficient value to maintain the desired objective. Further, if after reaching the target weight the subject regains weight, the amount of polypeptide conjugate can be increased, or if it is previously terminated, the administration can be restarted.
Similarly, in the area of glycemic control, the polypeptide conjugate can be administered according to the methods described herein for a period sufficient to achieve an objective HbA1c, a target fasting glucose level, a general target blood glucose concentration , etc., after which the plasma concentration of the polypeptide conjugate can be reduced to a maintenance level or discontinued. If discontinued, administration can be resumed later if necessary. In one embodiment, the polypeptide conjugate is administered according to the methods described herein for a period sufficient to reduce or stabilize fasting glucose levels, reduce or eliminate fasting glucose levels high or higher than is desired In some embodiments, the methods described herein also provide for the co-administration of the polypeptide conjugate with one or more antidiabetic agents and / or anti-obesity / appetite suppressant agents. By "co-administration" is meant the administration of two or more active agents in a single composition, the simultaneous administration in separate solutions, or alternatively they may be administered at different times from one another. Such antidiabetic agents include, without limitation, metformin, a sulfonylurea (SU), a thiazolidinedione (TZD) or any combination thereof. Exemplary agents include pioglitazone, rosiglitazone, glibenclamide, gliclazide, glimepiride, glipizide, gliquidone, chlorpropamide and tolbutamide. Additional agents include the inhibitors of dipeptidyl peptidase 4 (DPP-IV) such as vildagliptin or sitagliptin. The polypeptide conjugate can also be co-administered with insulin. Coadministration can be done by any suitable means or administration regime. Anti-obesity agents known or under investigation include appetite suppressants, including stimulants of the phenethylamine type, phentermine (optionally with fenfluramine or dexfenfluramine), diethylpropion, phendimetrazine, benzophetamine, sibutramine; nmonabant, other cannabinoid receptor antagonists; oxintomodulin; fluoxetine hydrochloride; Qnexa (topiramate and phentermine), bupropion and zonisamide, or bupropion and naltrexone; or lipase inhibitors, similar to xenical or Cetilistat or GT 389-255.
In one embodiment methods are provided for decreasing the frequency and / or severity of gastrointestinal effects, including nausea, or the number and / or severity of nausea events, associated with the administration of the polypeptide conjugate, comprising chronically administering a conjugate. of polypeptide by any of the methods described herein. Sometimes chronic administration starting with low or lower doses may induce tolerance to the administered polypeptide conjugate, such that high doses that normally cause a frequency and / or severity of unacceptable gastrointestinal effects, may be administered to the subject with reduced gastrointestinal effects. or absent. Thus, it is contemplated that chronic administration can be initiated with suboptimal dosing of the polypeptide conjugate using, for example, a formulation that releases the polypeptide conjugate administered during a period in which the formulation is administered weekly. Over a period of weeks, the plasma levels of the polypeptide conjugate will increase and eventually reach a plateau concentration. In some modalities this plateau is a concentration that could not be tolerated due to the gastrointestinal adverse effects if it was administered in a simple or initial dose. Any suitable prolonged release formulation and administration regimen can be used to achieve the plateau effect.
Accordingly, in one embodiment multiple doses of sustained release are provided, such that each successive dose increases the concentration of the agent or agents in the subject, where a therapeutically effective concentration of the agent or agents in the subject is reached. In a further embodiment, each successive sustained release dose is administered in such a manner that its sustained phase overlaps with the sustained phase of the previous dose.
The description also provides drug delivery devices having at least one therapeutically effective dose of the polypeptide conjugate described herein or the pharmaceutical composition containing the polypeptide conjugate described herein. The drug delivery devices can be single-use or multi-use bottles, single-use or multi-use pharmaceutical pens, single-use or multiple-use cartridges, etcetera. In one embodiment, the drug delivery devices contain the polypeptide conjugates or pharmaceutical compositions described herein in amounts capable of providing the subject from about 7 doses to about 40 doses, or sufficient doses to last about one week or about one week. month.
The description also provides a kit comprising a container comprising a polypeptide conjugate as described herein, optionally with instructions for the subject to use the polypeptide conjugate. The container may be a bottle, cartridge, ball-pen type device or other delivery device, for single use or for multiple use, as described herein.
The use of a conjugate is also specifically contemplated of polypeptide for the manufacture of a medicament for use in any of the methods of treatment described herein.
Additional modalities include the following: 1. - A polypeptide conjugate comprising the compound 1A or the compound 2A. 2. - The polypeptide conjugate of embodiment 1 comprising compound 1A. 3. - The polypeptide conjugate of embodiment 1 comprising compound 2A. 4. - The polypeptide conjugate of any of the modalities 1 to 3, wherein the compound 1A or the compound 2A is covalently linked to at least a portion of polyethylene glycol. 5. - The polypeptide conjugate of mode 4 wherein the polyethylene glycol is linked to a lipophilic moiety. 6. - The polypeptide conjugate of mode 5 wherein the lipophilic portion is an alkyl group, a fatty acid, a cholesteryl, or an adamantyl. 7. - The polypeptide conjugate compound of any of embodiments 1 to 3, wherein the compound 1A or the compound 2A is covalently linked to at least one fatty acid. 8. - The polypeptide conjugate compound of any of embodiments 1 to 3, wherein the compound 1A or the compound 2A is covalently bound to albumin. 9. - The polypeptide conjugate of mode 8 wherein the albumin is linked to a fatty acid. 10. - The polypeptide conjugate compound of any of embodiments 1 to 3, wherein the compound 1A or the compound 2A is covalently linked to a polyamino acid. 11. The polypeptide conjugate of any of embodiments 1 to 10, which has an EC50 less than 1 micromolar in a GLP-1 receptor function assay. 12. - The polypeptide conjugate of mode 11 having an EC50 less than 100 nanomolar in a function test of the receptor of GLP-1. 13. - The polypeptide conjugate of mode 12 having an EC50 less than 10 nanomolar in a function test of the GLP-1 receptor. 14. - The polypeptide conjugate of mode 13 having an EC50 less than 1 nanomolar in a function test of the GLP-1 receptor. 15. - The polypeptide conjugate of any of embodiments 1 to 14, wherein the polypeptide conjugate has an EC50 less than 1 micromolar in a calcitonin receptor function assay C1a. 16. The polypeptide conjugate of embodiment 15 wherein the polypeptide conjugate has an EC50 less than 100 nanomolar in a calcitonin receptor function assay C1a. 17. - The polypeptide conjugate of mode 16 wherein the polypeptide conjugate has an EC50 less than 10 nanomolar in a calcitonin receptor function assay C1a. 18. The polypeptide conjugate of embodiment 17 wherein the polypeptide conjugate has an EC50 less than 5 nanomolar in a calcitonin receptor function assay C1a. 19. The polypeptide conjugate of any of embodiments 1 to 18, wherein the polypeptide conjugate reduces body weight more potently than exendin 4, compound 4A or davalintide. 20. - The polypeptide conjugate of any of embodiments 1 to 19, wherein the polypeptide conjugate reduces body weight more potently than exendin 4 and davalintide. 21- The polypeptide conjugate of any of embodiments 1 to 20, wherein the polypeptide conjugate reduces body weight more efficiently than exendin 4. 22. - The polypeptide conjugate of any of embodiments 1 to 20, wherein the polypeptide conjugate reduces body weight more effectively than davalintide. 23. The polypeptide conjugate of any of embodiments 1 to 22, wherein the polypeptide conjugate reduces body weight more efficiently than exendin 4 and davalintide. 24. The polypeptide conjugate of any of embodiments 1 to 20, wherein the polypeptide conjugate reduces body weight more efficiently than co-administered maximum effective doses of exendin 4 and davalintide. 25. - The polypeptide conjugate of any of embodiments 1 to 24, wherein the polypeptide conjugate reduces body weight more potently and more efficiently than exendin 4. 26. - The polypeptide conjugate of any of embodiments 1 to 24, wherein the polypeptide conjugate reduces the body weight more potently and / or more efficiently than the compound 3A. 27. - The polypeptide conjugate of any of embodiments 1 to 26, wherein the reduction in body weight occurs over a period of 4 weeks. 28. - The polypeptide conjugate of any of embodiments 1 to 27, wherein the reduction in body weight occurs during a period of 6 months. 29. - The polypeptide conjugate of any of embodiments 1 to 28, wherein the reduction in body weight occurs over a period of 1 year. 30. - The polypeptide conjugate of any of embodiments 1 to 29, wherein the polypeptide conjugate produces reduced ingestion of kaolin in rats as compared to exendin 4. 31. - The polypeptide conjugate of any of the embodiments 1 to 30, wherein the polypeptide conjugate produces reduced ingestion of kaolin in rats as compared to davalintide. 32. The polypeptide conjugate of any of embodiments 1 to 31, wherein the polypeptide conjugate produces reduced ingestion of kaolin in the rats compared to compound 7A. 33. - The polypeptide conjugate of any of embodiments 1 to 32, wherein the polypeptide conjugate produces reduced nausea compared to exendin 4. 34. The polypeptide conjugate of any of embodiments 1 to 33, wherein the polypeptide conjugate produces reduced nausea compared to davalintide. 35. The polypeptide conjugate of any of embodiments 1 to 34, wherein the polypeptide conjugate produces reduced nausea compared to compound 7A. 36. - The polypeptide conjugate of any of embodiments 1 to 35, wherein the reduced nausea is less severe nausea or a lesser number of nausea adverse events per year, or both, wherein the nausea events may be mild, moderate or severe, or the combined total number of nausea events. 37. - The polypeptide conjugate of any of embodiments 1 to 36, wherein the polypeptide conjugate reduces fasting plasma glucose. 38. - The polypeptide conjugate of any of the embodiments 1 to 37, wherein the polypeptide conjugate increases tolerance to oral glucose loading. 39. - The polypeptide conjugate of any of embodiments 1 to 38, wherein the polypeptide conjugate increases insulin secretion induced by glucose. 40. The polypeptide conjugate of any of embodiments 1 to 39, wherein the polypeptide conjugate delays gastric emptying for at least two hours, at least four hours, or at least eight hours. 41. The polypeptide conjugate of any of embodiments 1 to 40, wherein the polypeptide conjugate delays gastric emptying to a greater degree than compound 3A at identical doses. 42. The polypeptide conjugate of any of embodiments 1 to 41, wherein the polypeptide conjugate reduces plasma triglycerides to a greater degree than exendin 4, davalintide or compound 3A at identical doses. 43. The polypeptide conjugate of any of embodiments 1 to 42, wherein the polypeptide conjugate reduces plasma triglycerides to a greater extent than compound 2A at identical doses. 44. The compound of any of clauses 1-43 in the form of a pharmaceutically acceptable salt. 45. - A pharmaceutical composition comprising a polypeptide conjugate according to any one of embodiments 1 to 44, and a pharmaceutically acceptable carrier. 46. A method of treating diabetes in a subject in need thereof or desirous thereof, comprising administering a therapeutically effective amount of the polypeptide conjugate or pharmaceutical composition of any of modalities 1-45, to treat diabetes in the subject . 47. - The method of clause 46, where diabetes is type 1 diabetes. 48. - The method of clause 46, where diabetes is type 2 diabetes. 49. - The method of clause 46, where diabetes is gestational diabetes. 50. - The method of any of the embodiments 46 to 49, wherein the subject is overweight, obese, or has the tendency to be overweight or obese. 51. - A method of treating insulin resistance in a subject in need thereof or desire thereof, which comprises administering a therapeutically effective amount of the polypeptide conjugate or pharmaceutical composition of any of clauses 1-45, to treat the resistance to insulin in the subject. 52. - A method of treating postprandial hyperglycemia in a subject in need thereof or desirous thereof, comprising administering a therapeutically effective amount of the polypeptide conjugate or pharmaceutical composition of any of Clauses 1-45, to treat postprandial hyperglycemia in the subject. 53. - A method for reducing the blood glucose levels of a subject in need of the same or desire thereof, which comprises administering a therapeutically effective amount of the polypeptide conjugate or pharmaceutical composition of any of clauses 1-45, to reduce the blood glucose levels of the subject. 54. A method for reducing HbA1c levels in a subject in need thereof, comprising administering a therapeutically effective amount of the polypeptide conjugate or pharmaceutical composition of any of clauses 1-45, to reduce HbA1c levels in the subject . 55. A method for stimulating the release of insulin in a subject in need thereof, comprising administering a therapeutically effective amount of the polypeptide conjugate or pharmaceutical composition of any of clauses 1-45, to stimulate the release of insulin in the subject . 56. A method for reducing gastric motility in a subject in need thereof, comprising administering a therapeutically effective amount of the polypeptide conjugate or pharmaceutical composition of any of clauses 1-45, to reduce gastric motility in the subject. 57. A method for delaying gastric emptying in a subject in need thereof, comprising administering a therapeutically effective amount of the polypeptide conjugate or pharmaceutical composition of any of clauses 1-45, to delay gastric emptying in the subject. 58. - A method for reducing food ingestion in a subject in need of the same or desire thereof, comprising administering a therapeutically effective amount of the polypeptide conjugate or pharmaceutical composition of any of clauses 1-45, to reduce ingestion of food in the subject. 59. - A method for reducing appetite in a subject in need of the same or desire thereof, comprising administering a therapeutically effective amount of the polypeptide conjugate or pharmaceutical composition of any of clauses 1-45, to reduce appetite in the subject. 60. A method for reducing weight in a subject in need of the same or desire thereof, comprising administering a therapeutically effective amount of the polypeptide conjugate or pharmaceutical composition of any of clauses 1-45, to reduce the weight in the subject. 61. A method of treating overweight in a subject in need thereof or desirous thereof, comprising administering a therapeutically effective amount of the polypeptide conjugate or pharmaceutical composition of any of clauses 1-45, to treat overweight in the subject. 62. A method of treating obesity in a subject in need thereof or desirous thereof, comprising administering a therapeutically effective amount of the polypeptide conjugate or pharmaceutical composition of any of clauses 1-45, to treat obesity in the subject . 63. - The method of any of clauses 46-62, wherein the therapeutically effective amount of the compound is from about 0.1 to about 5 mg. 64. - The method of any of clauses 46-62, wherein the therapeutically effective amount of the compound is from about 1 μg to about 2.5 mg. 65. - The method of any of clauses 46-62, wherein the therapeutically effective amount of the compound is from about 1 μg to about 1 mg. 66. - The method of any of clauses 46-62, wherein the therapeutically effective amount of the compound is from about 1 μg to about 50 μg. 67. - The method of any of clauses 46-62, wherein the therapeutically effective amount of the compound is from about 1 μg to about 25 μg. 68. - The method of any of clauses 46-62, wherein the therapeutically effective amount of the compound is from about 0.01 pg to about 100 pg, based on the weight of a subject of 70 kg. 69. The method of any of clauses 46-62, wherein the therapeutically effective amount of the compound is from about 0.01 pg to about 50 pg, based on the weight of a subject of 70 kg. 70. - A drug delivery device comprising at least one therapeutically effective dose of the compound or pharmaceutical composition of any of clauses 1-45. 71. - The drug delivery device of clause 70, wherein the drug delivery device is a bottle, a pharmaceutical pen, or a cartridge. 72. - The drug delivery device of clause 70 or 71, comprising a provision of therapeutically effective doses for about one month. 73. - A process for preparing the compound of any of the modalities 1 to 45. 74. - The modality 73 procedure, which comprises a recombinant process. 75. A kit comprising the polypeptide conjugate or pharmaceutical composition of any of embodiments 1 to 45, optionally having instructions for the use of the polypeptide conjugate or composition by the subject. 76. - The method of any of the above embodiments, wherein administration of the polypeptide conjugate results in improved therapeutic compliance of the patient compared to compound 6A, reduced reduction of severe hot flashes compared to compound 6A, and / or reduced nausea in comparison with compound 6A. 77. - The method of mode 63 where the dose is approximately 0.1 mg to 1.0 mg per day. 78. - The method of mode 67, where the dose is approximately 0.3 mg to 0.6 mg per day. 79. - The method of any of the above modalities where the dose is divided into two a day, or is given once a day.
EXAMPLES The following examples are for illustrative purposes only and are not considered to limit the scope of the claims.
EXAMPLE 1 Methods for in vivo studies in DIO rats The present study characterizes the metabolic actions of compound 1A and compound 2A. As described in the examples, the effect of 4 weeks of constant subcutaneous infusion of compound 1A and compound 2A (at 3, 10, 30 and 100 nmol / kg / d) was compared with the individual administration and the co-administration of the peptides of origin, compound 6A, compound 5 and compound 4A (at 2.8, 15 and 7.2 nmol / kg, maximum effective dose for weight loss) in male Sprague Dawley rats with diet-induced obesity (DIO). Several metabolic and PK parameters were evaluated.
Animals: Male Sprague Dawley rats (CRLCD rats, Charles River Laboratories, Wilmington, MA) were housed individually and maintained on a high-fat diet (32% kcal of fat; D12266B Research Diets, Brunswick, NJ) for approximately 8 weeks before the study. At the beginning of the tests (day 0) the average body weight of the rats was 545 ± 3.8 g.
Compounds: The compounds used in the examples include the following: Compound 4A, a C-terminally amidated full length exendin 4 peptide analog, with a single nucleotide difference at position 14 with respect to native exendin 4.
Compound 5, a chimera of the first 32 amino acids of exendin 4, which has amino acid substitutions at positions 14 and 28, followed by a 5 amino acid sequence of the C-terminus of a non-mammalian GLP-1 (frog).
Compound 6A, davalintide, a C-terminal amidated amylin mimetic; Compound 2A, a polypeptide conjugate of compound 5 covalently linked in frame to compound 6A through a glycine-glycine-glycine peptide linker; Y Compound 1A, a polypeptide conjugate of compound 4A covalently linked in frame to compound 6A through a glycine-glycine-glycine peptide linker.
Study design: Polypeptide conjugates were tested with respect to vehicle (50% DMSO in sterile water) and the compounds of origin alone and in combination (see Table 1). The doses of compound 5, compound 4A and compound 6A are maximum effective doses for weight loss in this model (data not shown). On day 1, the rats were surgically implanted with two osmotic minipumps (Alzet, Durect Corporation, Cupertino, CA) that delivered vehicle, compound 2A, compound 1A, compound 5, compound 4A or compound 6A, at a constant speed (nanomoles per kilogram of rat per day) for 4 weeks; see table 1.
TABLE 1 Assignment of groups and treatments Food intake and body weight were measured weekly. Body composition was evaluated on day -1 and day 28 using an NMR instrument (Echo Medical Systems, Houston, TX). The adiposity (percentage of fat mass) was defined as the amount of fat mass with respect to body weight (fat mass / body weight x 100). Blood was drawn from the tail vein on day 14. On day 28 a blood sample was taken through the jugular vein and the animals were euthanized by isoflurane overdose. The minipumps were immediately removed and the animals underwent a brief NMR scan, the tissues were collected for further histological examination and preliminary toxicological evaluation.
Statistical analysis: Data were analyzed using simple variance analysis (ANOVA) with post-hoc Newman-Keuls comparisons. Significance was assumed for p < 0.05. Graphs were generated using Prism 4 for Windows (Graphpad Software, San Diego, CA). All data points are expressed as mean ± SEM. For the highest doses of compound 1A and compound 2A, several of the animals had minimal food intake and these data were included in the analysis.
Analysis of hormones and metabolites. Plasma levels of the study drug were measured on day 14 and on completion by means of an ELISA. The percentage of hemoglobin A1c in whole blood (% HbA1c), plasma triglycerides, total cholesterol, HDL cholesterol and plasma glucose were measured on day 14 and on termination using an Olympus bioanalyzer (Olympus America Diagnostics). Plasma levels of insulin were analyzed on day 14 and on completion by means of an ELISA kit (Rat / mouse insulin ELISA, Lineo Diagnostics).
EXAMPLE 2 Compound 1A provides superior body weight loss Following the method of Example 1, each polypeptide conjugate (compound 1A and compound 2A) significantly reduced body weight, in a dose-dependent manner (p <0.05 with respect to the vehicle) at 3, 10, 30 and 100 nmol / kg / d during the 28-day treatment (see Figures 1A and 1B). Coadministration of the compounds of origin (compound 5 + compound 6A and compound 4A + compound 6A) significantly reduced body weight compared to vehicle controls and compared to compound 6A alone (p <0.05); see Figures 1A and 1B.
As indicated in Figure 1A, the corrected weight loss per vehicle at four weeks was: -31.5 ± 2.7% for compound 2A at 100 nmol / kg / d, -16.6 ± 3.6% for compound 5, -12.3 ± 1.3% for compound 6A, -24.3 ± 1.3% for compound 5 + compound 6A, p < 0.05 for compound 2A against compound 6A, but was not different from compound 5 or co-administration of compound 5 + compound 6A. For compound 2A there was no dose-effect beyond 10 nmol / kg / d; No further weight loss was observed at doses of 30 or 100 nmol / kg / d. A maximum weight loss was obtained in the group of 100 nmol / kg / d, at -27.8 ± 5.3%; see Figure A. Compound 2A was also several times more potent to reduce body weight by sustained infusion than compound 3A.
As indicated in Figure 1B, the corrected weight loss per vehicle for four weeks was: -37.3 ± 4.8% for compound 1A at 100 nmol / kg / d, -13.5 ± 1.5% for compound 4A, and -25.8 ± 1.5% for compound 4A + compound 6A; p < 0.05 for compound 1A against both peptides of origin alone or in combination. For compound A, dose-dependent weight loss was observed up to the highest dose tested [group of 100 nmol / kg / d at -37.3 ± 4.8%]. The weight loss with compound 1A at 100 nmol / kg / d was also significantly greater than the weight loss observed with the co-administration of maximum effective doses of the compounds of origin; see figure 2A. Although both polypeptide conjugates caused a very remarkable weight loss during the 28 day treatment period, for compound 1A the weight loss, even at the lowest dose tested, was significantly greater than with the individual administration of any of the origin peptides. Compound 1A was also markedly more potent and more effective than compound 2A. Also, compound 1A was approximately 10 times more potent to reduce body weight by sustained infusion than compound 3A.
EXAMPLE 3 Compound 1A provides a decrease in the intake of superior food Following the method of Example 1 as with body weight, each polypeptide conjugate significantly decreased food intake (p <0.05), at each dose tested, with respect to vehicle controls during the 28-day treatment period; see Figures 2A and 2B. At the highest dose tested, the total food intake was significantly reduced by compound 1A and compound 2A, with respect to the administration of a single peptide of origin; and at the highest dose of compound 1A, food ingestion was suppressed beyond the co-administration of compound 4A + compound 6A.
Cumulative feed intake of compound 2A at 3 nmol / kg / d was significantly higher than at the other doses (p <0.05). In comparison with the monotherapy with the compound of origin, the doses of 10 and 100 nmol / kg / day were significantly lower than any compound of origin; however, the dose of 30 nmol / kg / d alone was significantly different from the compound 6A of origin. The treatment with compound 5 + compound 6A co-administered was not significantly different from any of the doses of polypeptide conjugate; see figure 2A. Compound 2A was more potent for sharply reducing feed intake than compound 3A.
All doses of compound 1A significantly decreased food intake compared to the vehicle and any source compound administered alone; see Figure 2B. The highest dose of compound 1A was the only dose significantly different from the combination therapy (co-administration) of the two compounds of origin, compound 4A and compound 6A; see Figure 2B. Accordingly, compound 1A produced a greater decrease in food intake. Compound 1A was also more potent than compound 3A to sharply reduce food intake. In general, compound 1A and Compound 2A were more effective for weight loss with respect to the individual administration of the origin peptides in DIO rats, compound 1A exhibiting greater potency and efficacy for body weight loss compared to compound 2A.
EXAMPLE 4 Compound 1A provides an improvement in body composition Following the method of example 1, at the beginning of the study no significant differences were observed in the fat mass of the baseline, expressed in gross weight or as a percentage of fat mass (adiposity), or in lean mass of the baseline between subjects test (see table 2 and table 3). At termination it was observed that increasing doses of the polypeptide conjugate resulted in reductions in fat mass. The body weight loss induced by the polypeptide conjugate was associated with a significantly reduced percentage of fat mass (dose of 100 nmol / kg / d: -12.2 ± 1.3% for compound 2A, and -15.5 ± 2.2% for compound 1A; both p < 0.05 against vehicle controls). Compound 2A did not alter the percentage of lean mass; however, all doses of compound 1A increased the percentage of lean mass with respect to vehicle controls. Compound 1A and compound 2A at 100 nmol / kg / d reduced fat mass significantly compared to vehicle groups and administration of any parent compound (see Table 2 and Table 3). Compound 2A at 10 and 100 nmol / kg / d and the coadministration treatment of compound 5 + compound 6A significantly reduced terminal lean mass with respect to the vehicle (see Table 2). Treatment with compound 1A at 10, 30 and 100 nmol / kg / d, and treatment with compound 4A + compound 6A significantly reduced lean mass compared to vehicle controls (Table 3).
TABLE 2 Composition of the baseline and terminal body for compound 2A ' Groups that do not share a super index are significantly different from each other (p <0.05).
TABLE 3 Composition of the base line body v terminal for compound 1A \ Groups that do not share a super index are significantly different from each other (p <0.05).
To correct the loss of body weight, the fat and lean mass reductions were adjusted for the terminal body weight to calculate the change in adiposity (percentage of fat mass) and the change in the percentage of lean mass. Adiposity was significantly reduced with all doses of compound 2A and also with the compounds of origin alone and in combination (see Figure 3). Compound 2A at 100 nmol / kg / d produced the greatest decrease in adiposity (-12.2 ± 1.3%, Figure 3).
Reductions in adiposity were observed with compound 1A: all treatment doses significantly reduced adiposity compared to vehicle controls, as did administration of compound 4A and compound 6A; see Figure 4. The highest dose of compound A produced the highest reduction (-15.5 ± 2.2%), significantly lower than all other doses, but similar to the co-administration of the compounds of origin (see Figure 4) , and superior to compound 2A.
Although general lean (or fat-free) mass was reduced with high doses of the polypeptide conjugate, the change in lean mass, expressed as a percentage of body weight, was not significantly different between the vehicle control groups and any dose of compound 2A, compound 5, compound 6A, or compound 5 + compound 6A combined (see Figure 5). Surprisingly, treatment with all doses of compound 1A significantly increased the percentage of lean mass compared to vehicle controls (see Figure 6). Compound 1A is superior for improving the composition of the body, for example, saving lean mass and consuming fat mass.
At 28 days of exposure, Compound 1A and Compound 2A provide fasting blood glucose reduction (mg / dL), reduction of HbA1c and loss of body weight (consumption of fat mass and saving of lean mass) in mice ob / ob (obese and diabetic). In ob / ob mice, compound 1A produced a similar fasting blood glucose reduction loss with respect to exendin 4, but produced a superior weight loss (fat mass consumption and lean mass saving). These beneficial effects on the reduction of HbA1c and reduction of fasting blood glucose are even more surprising in view of the observed increase in HbA1c and the increase in fasting blood glucose observed with davalintide alone in this system. This superior effect of compound 1A is even more surprising compared to an increase in plasma glucose levels on day 28, observed in ob / ob mice treated with a combination of exendin 4 and davalintide (data not shown).
EXAMPLE 5 Compound 1A provides superior metabolic parameters Parameters in the plasma: The effects of the compounds of origin and the polypeptide conjugate on the following parameters in the plasma were determined: percentage of hemoglobin A1c (HbA1c), plasma insulin in the breakfast state, and the concentration of the polypeptide conjugate in the plasma, on days 14 and 28.
After 28 days of treatment with compound 2A no glucose change was observed, percentage of hemoglobin A1c (HbA1c), insulin, total cholesterol or HDL. Similarly, after 28 days there was no significant effect of compound 1A on total cholesterol or HDL or HbA1c levels. Plasma levels of insulin and glucose were significantly reduced by compound 1A at some doses, compared to vehicle controls. Plasma triglycerides were significantly reduced by all peptide treatments compared to the vehicle after 28 days of treatment. Plasma levels of compound 1A and compound 2A, measured by means of a specific immunoassay, were detected at increasing values corresponding to the treatment doses after 2 and 4 weeks of treatment.
After 14 days of treatment, the HbA1c levels were slightly but significantly elevated with the compound 5 + compound 6A, the compound 5, and the compound 2A, at 10 nmol / kg / d, with respect to the vehicle and vehicle groups. compound 6A (see table 4). After 28 days there were no apparent differences in HbA1c between any of these groups. All treatment groups exhibited significantly reduced insulin levels after 14 days with respect to vehicle controls; however, no differences were observed after 28 days. Plasma levels of compound 2A were significantly increased in plasma in a dose-dependent manner on days 14 and 28 (see table 4).
TABLE 4 Plasma levels of HbA1c, insulin and compound 2A 1. Groups that do not share a super-index are significantly different from each other.
In contrast, treatment with compound 1A at 3 and 10 nmol / kg / d increased HbA1 c levels compared to the vehicle and compound 6A groups after 14 days, with no differences observed at 28 days (see box 5). Similar to the treatment with compound 2A, all doses of compound 1A and the combination of compound 4A + compound 6A significantly reduced plasma insulin compared to the vehicle at 14 days, but were further reduced by treatment with the compound 1A to 10 and 30 nmol / kg / d and coadministration treatment of compound 4A + compound 6A, with respect to the vehicle after 28 days (see table 5). At 14 days compound 1A at 100 nmol / kg / d showed significantly higher plasma concentrations compared to the vehicle and compound 1A at 3 and 10 nmol / kg / d, but not at 30 nmol / kg / d. There were no differences between the values at 14 and 28 days.
TABLE 5 Plasma levels of HbA1c, insulin and compound 1A 1. Groups that do not share a superscript are significantly different from each other.
This study also evaluated plasma glucose levels and lipids (triglycerides, total cholesterol and HDL) after 28 days. No dose of compound 2A and of the source peptides administered separately or together, alter glucose or total cholesterol or HDL. Triglycerides were significantly reduced in all groups compared to the vehicle (see Table 6).
TABLE 6 Glucose and plasma lipids: Compound 2A In contrast to all other compounds, including compound 2A, compound 1A reduced HDL cholesterol to 10 and 30 nmol / kg / d, for example in comparison to compound 6A. Compound 1A does not alter total cholesterol; see Table 7. All doses of compound 1A and the origin peptides reduced triglycerides relative to the vehicle, and reduced glucose to 10 and 30 nmol / kg / d, same as compound 4A + compound 6A (see table 7).
TABLE 7 Glucose and plasma lipids: Compound 1A EXAMPLE 6 Polypeptide conjugates retain double receptor agonism wanted The polypeptide conjugates, compound 1A, compound 2A, compound 3A, and the compounds of origin, were tested in a cell-based assay for their ability to bind and exhibit agonism with the GLP-1 receptor (e.g., similar activity of exendin 4), or the calcitonin receptor (e.g., davalintide / amylin mimetic mimetic activity).
The functional assay of the GLP-1 receptor measures the cAMP increases in the cell line 6-23 (clone 6) (Zeytinoglu et al. "Establishment of a calcitonin-producing rat medullary thyroid carcinoma cell line: I. Morphological studies of the tumor and cells in culture ", Endocrinology 107: 509-515 (1980); Crespel et al.," Effects of glucagon and glucagon-like peptide-1- (7-36) amide on C cells from thyroid and medullary thyroid cancer carcinoma CA-77 cell line ", Endocrinology 37: 3674-3680 (1996)) by activation induced by endogenously expressed GLP-1 receptor peptide. Accumulation of cAMP is measured after 30 minutes of peptide treatment using the HTRF cell-based cAMP assay kit (CisBio, Bedford, MA, USA) in 384-well format. The HTRF (homogeneous fluorescence resolved with time) is a technology based on TR-FRET (time-resolved fluorescence and energy transfer by fluorescence resonance), a combination of FRET chemistry and the use of fluorophores with long half-lives of emission . The efficacy of the test compound is determined with respect to the treatment of the cell with forskolin 10 microM (a constitutive activator of adenylate cyclase that generates cAMP), and the potency (EC50) of the test compound is determined by the analysis of a test curve. concentration-response using a non-linear regression analysis fitted to a 4-parameter model. The concentration-response curves vary in concentrations of the test compound from 1 micromolar to 0.1 picomolar (n = 4 duplicates per concentration). The test compounds are diluted 1: 10 in series for an eight-point dose-response. The cells are suspend at 2.5 x 10 6 cells / ml in buffer containing IB 500 microM and HTRF solution, Five microliters of the test compound are added to 5 microliters of suspended cells and incubated for 30 minutes in the dark at room temperature. Activation is stopped by adding detection reagent / lysis buffer The accumulated AMPc was determined according to the kit instructions.
The functional activity of the test compounds in the calcitonin C1a receptor was determined by accumulation of cAMP in a C1a-HEK cell line overexpressing the rat C1a calcitonin receptor, after a 30 minute exposure to the test compound, using the HTRF-based cAMP assay kit (CisBio, Bedford, MA USA) in a 384-well format. Accumulation of cAMP is measured in response to increasing concentrations of test peptide and the efficacy of said peptide is determined with respect to treatment of the cell with forskolin 10 microM (a constitutive activator of adenylate cyclase). The functional activity of a test compound can also be measured using an AlphaScreen full-cell cAMP functional assay (Perkin Elmer, MA, USA). The calculated EC50 values are based on 4-parameter concentration-response curves with peptide doses at concentrations of 1 micromolar to 0.1 picomolar (n = 4 duplicates per concentration). The cells are suspended at 2.0 x 10"6 cells / ml in buffer containing IBMX 500 microM and HTRF solution, Five microliters of the test compound are added to 5 microliters of suspended cells and incubated for 30 minutes in the dark at room temperature. The activation is stopped by adding detection reagent / lysis buffer The accumulated AMPc was determined according to the kit instructions.
Table 8 provides EC50 measurements for cAMP, an in vitro indicator of receptor activity. As expected, exendin 4, compound 4A, compound 5 and compound 10A, all GLP-1 receptor agonists, were active in the GLP-1 receptor function assay, but not in the GLP-1 receptor assay. calcitonin C1 receptor function a. Exendin 4 was more active than any of the other compounds of origin. As expected, compound 6A, a mimetic amylin davalintide, was active in the calcitonin receptor function assay C1 a but not in the GLP-1 receptor function assay.
TABLE 8 Receiver agonism Function Function No. of GLP-1 (nM) calcitonin compound (nM) Comp. 6A inactive 0.05-0.11 Exendina 4 0.004 inactive Comp. 5 0.04 inactive Comp. 4A 0.05 inactive Comp. 10A 0.09 inactive Comp. 3A 0.05 2.1 Comp. 2A 0.36 1.8 Comp. 1A 0.19 3.2 In addition, compound 1A and compound 2A in vivo have amylin mimetic active sequences based on a demonstrated effect of each compound to reduce calcium in the blood, a property of amylin agonists, but not of exendin agonists (data not shown). Accordingly, all of the conjugated compounds, compound 1A, compound 2A and compound 3A (a previously known conjugate) were functional at the GLP-1 and amylin receptors.
EXAMPLE 7 Polypeptide conjugates are active to reduce basal glucose The polypeptide conjugates and the compounds of origin were analyzed in a basal glucose reduction assay in vivo. This assay reflects the ability of the conjugated polypeptide to increase insulin-mediated glucose clearance in the orally administered glucose test (OGTT). OGTT is used to diagnose diabetes, although the simplest fasting plasma glucose test that measures the subject's plasma glucose level after a fast of at least eight hours is preferred. The following procedure was used: The test compound was injected at various concentrations intraperitoneally (i.p.) to NIH / Swiss female mice with fasting from t = -5 min to 4 h. For forced feeding they were given glucose (1.5 g / kg) at t = 0. Samples were taken at t = 30 min as blood glucose from the tail using a OneTouch® Ultra® (LifeScan, Inc., Milpitas, CA). Significant effects were identified by ANOVA (p <; 0.05), followed by a Dunnett post test using GraphPad Prism version 4.00 for Windows (GraphPad Software, San Diego CA).
The results are presented in Table 9. As expected, exendin 4 and the peptide analogs of exendin 4, compound 4A, compound 5 and compound 10A, are active to reduce glucose. Compound 2A is surprisingly more potent than any of the previously known polypeptide conjugates, compound 3A and compound 7A. Compound 1A is surprisingly more potent than Compound 2A and also Compound 3A and Compound 7A.
OGTT EXAMPLE 8 The conjugates of polypeptide compound 1A and compound 2A reduce the ingestion of food with less nausea To investigate the possible effects of nausea of the conjugated polypeptide, the acute ingestion of kaolin in the rats was measured. Pica conduct (dirt / clay intake) is a marker of nausea in rodents, usually associated with a decrease in food intake and weight loss. The pike can be evaluated by measuring the ingestion of kaolin synthetic clay. Cisplatin, a chemotherapeutic drug that can act in the intestine to produce emesis, was used as a positive control to induce hypophagia associated with nausea. The rats were acclimated to kaolin for 3 days as kaolin clay mixed with regular food. Then the baseline ingestion of kaolin and food was measured at 4 h and 24 h. Subsequently, the rats were fasted for approximately 16 h, after which the test compound was injected at the dose indicated below. Twenty-four hours after the injection, the consumption of food and kaolin was measured. Table 10 presents the results of the ingestion of kaolin and the correlation with the inhibition of the ingestion of food (food).
TABLE 10 Nausea Compound 7A, a previously known conjugate, at a dose that inhibited food ingestion, induced a significant kaolin ingestion similar to the positive control, a sign of nausea in the rats. Compound 3A at doses that suppress acute food intake similar to the cisplatin injection had only a modest effect, if any, on the consumption of kaolin. Compound 1A and compound 2A, at doses that cause reduction in feed intake, even greater than the positive control, compound 3A or compound 7A, do not induce significant increases in the consumption of kaolin. Surprisingly, in spite of the nausea associated with exendin 4 and davalintide, compound 1A and compound 2A did not produce significant ingestion of kaolin in this study.
As shown, compound 1A shows a high potency for the GLP-1 receptor and the amylin and calcitonin receptors, demonstrating that its portions of davalintide and exendin-like retain their biological activity. The activities for these target receptors are only moderately attenuated compared to the compounds of origin. Interestingly, compound 1A binds to the CGRP receptor with very low affinity, exhibiting better selectivity than compound 3A for the amylin receptor (> 600 fold vs.> 100 fold, respectively), and calcitonin receptors (> 1600 times vs.> 280 times) against the CGRP receptor, and even better than the selectivity of davalintide (compound 6A) for binding to the calcitonin and amylin receptors against the CGRP receptor. Although davalintide is a potent antagonist of the adrenomedullin receptor (Cl50 = 18 nM), compound 1A does not exhibit functional activation or antagonism of the adrenomedullin receptor at concentrations up to 10 uM. Accordingly, compound 1A exhibits a surprisingly different pharmacological profile compared to davalintide, with respect to cellular receptors that recognize amylin and amylinimimetics. Compound 1A has fewer non-target activities than the origin peptide. It is expected that this improved pharmacological profile of compound 1A will result in fewer side effects, such as reduced incidence of severe hot flashes, nausea and / or vomiting, particularly with human subjects, compared to the parent peptide, compound 6A. For example, CGRP and CGRP agonists have been reported to induce severe hot flashes and even nausea and vomiting in human subjects, which are believed to be due in part to the activation of CGRP receptors and relieved by CGRP antagonists.
It is expected that the compound 1A and the compound 2A, and particularly the compound 1A, have a greater therapeutic compliance on the part of the patient, and / or will allow to increase the dose as necessary, in comparison with the previous compounds, for example in comparison with Compound 6A, resulting in better commercial success.
EXAMPLE 9 Polypeptide conjugates delay gastric emptying The polypeptide conjugates and the compounds of origin were analyzed for their ability to delay gastric emptying in the rats. Inhibition of gastric emptying is a physiological effect of GLP-1 receptor agonism and also of amylin receptor agonism, and a key pharmacological effect of exendin 4 and compound 6A on glucose control. Male Sprague Dawley rats (-250 grams, n = 5 per group) received fasting a single subcutaneous injection of saline, compound 6A or test compound at t = 0 (1 nmol / g). The rats then received oral forced feeding of 33 mg paracetamol / 1 ml Orablend (Paddock Laboratories, Inc., MN, USA) at t = 3.5 h, 5.5 h or 7.5 h after injection. Blood was drawn to measure paracetamol at 4 h, 6 h or 8 h after injection s.c. Gastric emptying was evaluated by the appearance of paracetamol in plasma 30 min after oral forced feeding.
Table 1 1 presents the percentage of inhibition of gastric emptying. Compound 1A and compound 2A were as effective as compound 6A in inhibiting gastric emptying up to six hours after a single injection. Compound 3A and compound 7A do not significantly inhibit gastric emptying at the time point and doses tested. Surprisingly, compound 1A showed a longer duration of action compared to compound 2A.
TABLE 11 Delayed gastric emptying The effect of compound 1A, a polypeptide conjugate comprising the parent compound, compound 4A, in the assays provided herein, is surprisingly. Hargrove et al., 2007, teaches that compound 4A, the peptide analogue of exendin 4 Leu14-exendin 4 produces a markedly less potent gastric emptying delay (4 times lower), a markedly less potent food ingestion inhibition ( 8 times smaller), a shorter half-life and a shorter duration of action compared to exendin 4. Despite the presence of the peptide analog sequence Leu14-exendin 4 in the conjugate of polypeptide compound 1A, the compound 1A exhibits surprisingly superior pharmacological properties compared to compound 2A and previously known conjugates, such as robust and prolonged action of gastric emptying, and also surprisingly robust and prolonged action of reducing food intake and reducing body weight. Surprisingly, compound 1A and compound 2A have a more stable metabolic profile (at least twice) in vitro in human plasma and with human kidney brush border membrane matrices during an incubation period of 5 h, in comparison with exendin 4, compound 4A and compound 6A (data not shown). No metabolites were detected for compound 1A or compound 2A during that period, indicating that any unidentified metabolite could be present at levels below 0%.
Surprisingly, compound 1A and compound 2A have a longer half-life with similar bioavailability (subcutaneous injection in the rats) compared to exendin 4, compound 4A or compound 6A (data not shown). Compound 1A has a half-life of 72 minutes administered subcutaneously (in male Sprague-Dawley rats), compared to 30 minutes of exendin 4, and approximately 30 minutes of davalintide, with similar absolute bioavailability.
These superior pharmacological properties, associated with an excellent PK profile and other favorable pharmacological properties, such as less off-target activity and reduced nausea, provide a surprisingly useful polypeptide conjugate (compound 2A and even more compound A), to control glucose with improvement of the control of weight and body composition in the subjects in need of said treatment, who have diseases or conditions where such treatment is beneficial. These conditions include prediabetes, diabetes, overweight or obese diabetes, overweight or obesity, wherein said subjects have a need or desire to control their blood glucose (e.g., anti-hyperglycemia) and / or have an improved effect or control of your body weight or body composition to reduce body weight, maintain body weight, prevent an increase in body weight, and / or improve the ratio of lean muscle to body fat.
All publications and patent applications are incorporated herein by reference and to the same extent as if it were specifically and individually indicated to be incorporated as a reference, and as if fully indicated herein. Although the foregoing has been described in detail for purposes of clarity of understanding, it will be apparent to the person skilled in the art that changes and modifications can be made without departing from the spirit or scope of the appended description or claims.

Claims (79)

NOVELTY OF THE INVENTION CLAIMS
1. - A polypeptide conjugate comprising the compound 1A or compound 2A.
2. - The polypeptide conjugate according to claim 1, further characterized in that it comprises compound 1A.
3. - The polypeptide conjugate according to claim 1, further characterized in that it comprises compound 2A.
4. - The polypeptide conjugate according to any of claims 1 to 3, further characterized in that the compound 1A or the compound 2A are covalently linked to at least a portion of polyethylene glycol.
5. - The polypeptide conjugate according to claim 4, further characterized in that the polyethylene glycol is linked to a lipophilic portion.
6. The polypeptide conjugate according to claim 5, further characterized in that the lipophilic portion is an alkyl group, a fatty acid, a cholesteryl, or an adamantyl.
7. - The polypeptide conjugate compound according to any of claims 1 to 3, further characterized in that the compound 1A or the compound 2A are covalently linked to minus a fatty acid.
8. The conjugate polypeptide compound according to any of claims 1 to 3, further characterized in that the compound 1A or the compound 2A are covalently bound to albumin.
9. - The polypeptide conjugate according to claim 8, further characterized in that the albumin is bound to a fatty acid.
10 -. 10 - The polypeptide conjugate compound according to any of claims 1 to 3, further characterized in that the compound 1A or the compound 2A are covalently linked to a polyamino acid.
11. - The polypeptide conjugate according to any of claims 1 to 10, further characterized in that it has an EC50 less than 1 micromolar in a function test of the GLP-1 receptor.
12. The polypeptide conjugate according to claim 11, further characterized in that it has an EC50 less than 100 nanomolar in a GLP-1 receptor function assay.
13. - The polypeptide conjugate according to claim 12, further characterized in that it has an EC50 of less than 10 nanomolar in a function test of the GLP-1 receptor.
14. The polypeptide conjugate according to claim 13, further characterized in that it has an EC50 less than 1 nanomolar in a GLP-1 receptor function assay.
15. The polypeptide conjugate according to any of claims 1 to 14, further characterized in that it has an EC50 less than 1 micromolar in a calcitonin receptor function assay C1a.
16 -. 16 - The polypeptide conjugate according to claim 15, further characterized in that it has an EC50 less than 100 nanomolar in a calcitonin receptor function assay C1a.
17 -. 17 - The polypeptide conjugate according to claim 16, further characterized in that it has an EC50 less than 10 nanomolar in a calcitonin receptor function assay C1a.
18. The polypeptide conjugate according to claim 17, further characterized in that it has an EC50 less than 5 nanomolar in a calcitonin receptor function assay C1a.
19 -. 19 - The polypeptide conjugate according to any of claims 1 to 18, further characterized in that it reduces body weight more potently than exendin 4, compound 4A or davalintide.
20. The polypeptide conjugate according to any of claims 1 to 19, further characterized in that it reduces body weight more potently than exendin 4 and davalintide.
21. The polypeptide conjugate according to any of claims 1 to 20, further characterized in that it reduces body weight more efficiently than exendin 4.
22 -. 22 - The polypeptide conjugate according to any of claims 1 to 20, further characterized in that it reduces body weight more effectively than davalintide.
23. The polypeptide conjugate according to any of claims 1 to 22, further characterized in that it reduces body weight more efficiently than exendin 4 and davalintide.
24 -. 24 - The polypeptide conjugate according to any of claims 1 to 20, further characterized in that it reduces the body weight more effectively than co-administered maximum effective doses of exendin 4 and davalintide.
25. The polypeptide conjugate according to any of claims 1 to 24, further characterized in that it reduces body weight more potently and more efficiently than exendin 4.
26. The polypeptide conjugate according to any of claims 1 to 24, further characterized in that it reduces the body weight more potently and / or more efficiently than the compound 3A.
27. - The polypeptide conjugate according to any of claims 1 to 26, further characterized in that the reduction in body weight occurs during a period of 4 weeks.
28. - The polypeptide conjugate according to any of claims 1 to 27, further characterized in that the reduction in body weight occurs during a period of 6 months.
29. - The polypeptide conjugate according to any of claims 1 to 28, further characterized in that the reduction in body weight occurs during a period of 1 year.
30. - The polypeptide conjugate according to any of claims 1 to 29, further characterized in that it produces reduced ingestion of kaolin in rats compared to exendin 4.
31. The polypeptide conjugate according to any of claims 1 to 30, further characterized in that it produces reduced ingestion of kaolin in rats as compared to davalintide.
32. The polypeptide conjugate according to any of claims 1 to 31, further characterized in that it produces reduced ingestion of kaolin in the rats compared to compound 7A.
33. The polypeptide conjugate according to any of claims 1 to 32, further characterized in that it produces reduced nausea compared to exendin 4.
34. - The polypeptide conjugate according to any of claims 1 to 33, further characterized by producing reduced nausea compared to davalintide.
35. The polypeptide conjugate according to any of claims 1 to 34, further characterized in that it produces reduced nausea compared to compound 7A.
36. - The polypeptide conjugate according to any of claims 1 to 35, further characterized in that the reduced nausea is a less severe nausea or a lower number of adverse events of nausea per year, or both, wherein the nausea events may be mild, moderate or severe or the combined total number of nausea events.
37. - The polypeptide conjugate according to any of claims 1 to 36, further characterized in that it reduces fasting plasma glucose.
38 -. 38 - The polypeptide conjugate according to any of claims 1 to 37, further characterized by increasing tolerance to oral glucose loading.
39. - The polypeptide conjugate according to any of claims 1 to 38, further characterized in that it increases insulin secretion induced by glucose.
40. The polypeptide conjugate according to any of claims 1 to 39, further characterized in that it delays gastric emptying for at least two hours, at least four hours, or at least eight hours.
41 -. 41 - The polypeptide conjugate according to any of claims 1 to 40, further characterized in that it delays gastric emptying to a greater degree than compound 3A at identical doses.
42. The polypeptide conjugate according to any of claims 1 to 41, further characterized in that it reduces plasma triglycerides to a greater degree than exendin 4, davalintide or compound 3A at identical doses.
43. The polypeptide conjugate according to any of claims 1 to 42, further characterized in that it reduces the plasma triglycerides to a greater degree than the compound 2A at identical doses.
44. - The polypeptide conjugate according to any of claims 1-43 in the form of a pharmaceutically acceptable salt.
45. - A pharmaceutical composition comprising a polypeptide conjugate as claimed in any of claims 1 to 44, and a pharmaceutically acceptable carrier.
46. - The use of the polypeptide conjugate of any of claims 1-44 or pharmaceutical composition of claim 45, in the manufacture of a medicament for treating diabetes in a subject.
47. - The use as claimed in claim 46, wherein diabetes is type 1 diabetes.
48. - The use as claimed in claim 46, wherein the diabetes is type 2 diabetes.
49. - The use as claimed in claim 46, wherein diabetes is gestational diabetes.
50. - The use as claimed in any of claims 46 to 49, wherein the subject is overweight, obese, or has the tendency to be overweight or obese.
51. - The use of the polypeptide conjugate of any of claims 1-44 or pharmaceutical composition of claim 45, in the manufacture of a medicament for treating insulin resistance in a subject.
52 -. 52 - The use of the polypeptide conjugate of any of claims 1-44 or pharmaceutical composition of claim 45, in the manufacture of a medicament for treating postprandial hyperglycemia in a subject.
53. - The use of the polypeptide conjugate of any of claims 1-44 or pharmaceutical composition of claim 45, in the manufacture of a medicament for reducing the blood glucose levels of a subject.
54. - The use of the polypeptide conjugate of any of claims 1-44 or pharmaceutical composition of claim 45, in the manufacture of a medicament for reducing HbA1c levels in a subject.
55. - The use of the polypeptide conjugate of any of claims 1-44 or pharmaceutical composition of claim 45, in the manufacture of a medicament for stimulating the release of insulin in a subject.
56. - The use of the polypeptide conjugate of any of claims 1-44 or pharmaceutical composition of claim 45, in the manufacture of a medicament for reducing gastric motility in the subject.
57. - The use of the polypeptide conjugate of any of claims 1-44 or pharmaceutical composition of claim 45, in the manufacture of a medicament for delaying gastric emptying in the subject.
58. - The use of the polypeptide conjugate of any of claims 1-44 or pharmaceutical composition of claim 45, in the manufacture of a medicament for reducing the food intake in the subject.
59. - The use of the polypeptide conjugate of any of claims 1-44 or pharmaceutical composition of claim 45, in the manufacture of a medicament for reducing appetite in the subject.
60. - The use of the polypeptide conjugate of any of claims 1-44 or pharmaceutical composition of claim 45, in the manufacture of a medicament for reducing weight in the subject.
61. - The use of the polypeptide conjugate of any of claims 1-44 or pharmaceutical composition of claim 45, in the manufacture of a medicament for treating overweight in the subject.
62. - The use of the polypeptide conjugate of any of claims 1-44 or pharmaceutical composition of claim 45, in the manufacture of a medicament for treating obesity in the subject.
63. - Use as claimed in any of the claims 46-62, wherein the polypeptide conjugate is present in an amount of about 0.1 μg to about 5 mg.
64. - The use as claimed in any of claims 46-62, wherein the polypeptide conjugate is present in an amount of about 1 μg to about 2.5 mg.
65. - The use as claimed in any of claims 46-62, wherein the polypeptide conjugate is present in an amount of about 1 μg to about 1 mg.
66. - The use as claimed in any of claims 46-62, wherein the polypeptide conjugate is present in an amount of about 1 μg to about 50 μg.
67. - The use as claimed in any of claims 46-62, wherein the polypeptide conjugate is present in an amount of about 1 μg to about 25 μg.
68 -. 68. The use as claimed in any of claims 46-62, wherein the polypeptide conjugate is present in an amount of about 0.01 pg to about 100 pg, based on the weight of a subject of 70 kg.
69. - The use as claimed in any of claims 46-62, wherein the polypeptide conjugate is present in an amount of about 0.01 pg to about 50 pg, based on the weight of a subject of 70 kg.
70. - A drug delivery ce comprising at least one therapeutically effective dose of the polypeptide conjugate of any of claims 1-44 or pharmaceutical composition of claim 45.
71. - The drug delivery ce according to claim 70, further characterized in that the drug delivery ce is a bottle, a pharmaceutical pen, or a cartridge.
72. - The drug delivery ce according to claim 70 or 71, further characterized in that it comprises a provision of therapeutically effective doses for about one month.
73. - A method for preparing the polypeptide conjugate of any of claims 1 to 44.
74. - The method according to claim 73, further characterized in that it comprises a recombinant process.
75. - A kit comprising the polypeptide conjugate of any of claims 1-44 or pharmaceutical composition of claim 45, optionally having instructions for the use of the polypeptide conjugate or composition by the subject.
76. - The use as claimed in any of claims 46-69, wherein the polypeptide conjugate is adapted to result in an improved therapeutic compliance of the patient compared to compound 6A, less reduction of severe hot flashes compared to the compound 6A, and / or reduced nausea compared to the compound 6A.
77. - The use as claimed in claim 63, wherein the dose is approximately 0.1 mg to 1.0 mg per day.
78. - The use as claimed in claim 67, wherein the dose is approximately 0.3 mg to 0.6 mg per day.
79 -. 79 - The use as claimed in any of claims 77 and 78, wherein the dose is adapted to be administrable once a day or divided into two a day.
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